Antibiotic susceptibility of microorganisms and related methods and systems

ABSTRACT

Provided herein is an antibiotic susceptibility test and related compositions, methods and systems based on detection of a nucleic acid from a target microorganism in a sample in the presence or absence of a lysis treatment of the sample.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional Application No. 62/968,735, entitled “Accessibility-Enhanced Rapid Antibiotic Susceptibility Testing” filed on Jan. 31, 2020, with docket number P2456-USP2, and is also a continuation-in-part of the U.S. application Ser. No. 16/158,233, entitled “Antibiotic Susceptibility of Microorganisms and Related Compositions, Methods and Systems” filed on Oct. 11, 2018, with docket number P2283-US which in turn claims priority to U.S. Provisional Application No. 62/571,128, entitled “Antibiotic Susceptibility Testing (AST) Via Measurement of Nucleic Acid Accessibility” filed on Oct. 11, 2017, with docket number CIT 7880-P, and to U.S. Provisional Application No. 62/722,124, entitled “Antibiotic Susceptibility Testing (AST) Via Measurement of Nucleic Acid Accessibility” filed on Aug. 23, 2018, the contents of each of which is incorporated by reference in its entirety. the contents of each of which is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT GRANT

This invention was made with government support under Grant No. HR0011-11-2-0006 awarded by DARPA, Grant No. EB012946 and Grant No. GM007616 awarded by the National Institutes of Health, Grant No. IDSEP160030-02 awarded by the Department of Health and Human Services (HHS) Office of the Assistant Secretary for Preparedness and Response (ASPR) and the Wellcome Trust under the CARB-X program, Grant No. MCDC-18-01-01-007 awarded by the Defense Threat Reduction Agency (DTRA); an effort sponsored by the U.S. Government under Other Transaction number W15QKN-16-9-1002 between the MCDC, and the Government. The government has certain rights in the invention.

FIELD

The present disclosure relates to microorganisms and related biology as well as to diagnosis and treatment of related conditions in individuals. In particular, the present disclosure relates to antibiotic susceptibility of microorganisms and related markers, compositions, methods and systems.

BACKGROUND

Antibiotic susceptibility is an important feature of the biology of various microorganisms, which can be used in identifying approaches to treat or prevent bacterial infections.

Ideal antibiotic therapy is based on determination of the etiological agent for a particular condition and determination of the antibiotic sensitivity of the identified agent. In particular, the effectiveness of individual antibiotics varies with various factors including the ability of the microorganism to resist or inactivate the antibiotic.

Despite progress in identifying methods and systems to test antibiotic susceptibility for various microorganisms, as well as the identification of related markers, determination of antibiotic susceptibility can still be challenging, in particular, determination of antibiotic susceptibility when a rapid and accurate detection is desired for microorganisms such as Neisseria gonorrhoeae which are slow growing and lack the classic transcriptional SOS response to DNA damage.

SUMMARY

Provided herein is an antibiotic susceptibility test (sometimes abbreviated as AST) and related compositions, methods and systems based on nucleic acid detection which in several embodiments allows determination of antibiotic susceptibility of microorganisms as well as the diagnosis and/or treatment of related infections in individuals.

According to a first aspect, a method is described to detect a nucleic acid of a microorganism in a sample comprising the microorganism. The method comprises detecting in an antibiotic treated sample a nucleic acid of the microorganism, the detecting performed by

quantitatively detecting in the antibiotic treated sample a nucleic acid of the microorganism, the detecting performed

in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; or

-   -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of nucleic acid from the         microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of microorganism from the nucleic         acid in the antibiotic treated sample,         to obtain a detected antibiotic treated nucleic acid         concentration value of the microorganism in the antibiotic         treated sample. to obtain a detected nucleic acid in an         antibiotic treated sample.

According to a second aspect, a method to perform an antibiotic susceptibility test for a microorganism is described. The method comprises detecting susceptibility to an antibiotic of the microorganism, by

quantitatively detecting in a sample comprising the microorganism a nucleic acid of the microorganism following contacting the sample with the antibiotic:

in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; or

-   -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of nucleic acid from the         microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample following pre-lysis separation of microorganism from the         nucleic acid in the antibiotic treated sample.

According to a third aspect, a method is described to detect a marker of susceptibility to an antibiotic in a microorganism. The method comprises contacting with the antibiotic a sample comprising the microorganism, the contacting performed

in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; or

-   -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of nucleic acid from the         microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of microorganism from the nucleic         acid in the antibiotic treated sample,         to obtain an antibiotic treated sample.         The method further comprises quantitatively detecting in the         antibiotic treated sample a nucleic acid of the microorganism to         obtain an antibiotic treated concentration value,         The method also comprises detecting a nucleic acid concentration         ratio in the sample by comparing the detected antibiotic treated         nucleic acid concentration value with a detected reference         nucleic acid concentration value of the nucleic acid of the         microorganism in the sample, the nucleic acid concentration         ratio is indicative of resistance or susceptibility of the         microorganism to the antibiotic.

According to a fourth aspect, a method to diagnose susceptibility to an antibiotic of a microorganism infection in an individual is described. The method comprises contacting with the antibiotic a sample from the individual, the sample including the microorganism, the contacting performed

in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; or

-   -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of nucleic acid from the         microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of microorganism from the nucleic         acid in the antibiotic treated sample,         to obtain an antibiotic treated sample.         The method further comprises detecting in the antibiotic treated         sample a nucleic acid of the microorganism to obtain a detected         antibiotic treated concentration value.         The method also comprises detecting a nucleic acid concentration         ratio in the sample of the individual by comparing the detected         antibiotic treated nucleic acid concentration value with a         detected reference nucleic acid concentration value of the         nucleic acid of the microorganism in the sample of the         individual, and comparing the detected nucleic acid         concentration ratio in the sample with a threshold to diagnose         the antibiotic susceptibility of the microorganism infection in         the individual.

According to a fifth aspect, a method is described to detect antibiotic susceptibility of a microorganism and treat an infection of the microorganism in an individual. The method comprises contacting a sample from the individual with an antibiotic, the contacting performed

-   -   in absence of a lysis treatment of the antibiotic treated sample         targeting the microorganism; or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of nucleic acid from the         microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         following pre-lysis separation of microorganism from the nucleic         acid in the antibiotic treated sample,         to obtain an antibiotic treated sample.         The method further comprises detecting in the antibiotic treated         sample, a nucleic acid of the microorganism to obtain an         antibiotic treated concentration value.         The method also comprises detecting a nucleic acid concentration         ratio in the sample of the individual by comparing the detected         antibiotic treated nucleic acid concentration value with a         detected reference nucleic acid concentration value of the         nucleic acid of the microorganism in the sample of the         individual.         The method further comprises diagnosing antibiotic         susceptibility of the microorganism infection in the individual         by comparing the nucleic acid concentration ratio with a         threshold.         The method additionally comprises administering an effective         amount of the antibiotic to an individual diagnosed with a         microorganism susceptible to the antibiotic.

According to a sixth aspect, a system is described for performing at least one of the methods herein described to detect a nucleic acid of a microorganism in a sample, to detect antibiotic susceptibility of a microorganism, to perform an antibiotic susceptibility test for the microorganism, and/or to diagnose and/or treat a microorganism infection in an individual. The system comprises an antibiotic, at least a probe specific for a nucleic acid of the microorganism or for a polynucleotide complementary thereto, and reagents for detecting the at least one probe. The system can optionally comprise reagents to perform a lysis treatment, a separation treatment and/or mechanical separation of the sample for concurrent sequential or combined use in any one of the methods of the disclosure.

According to a seven aspect, any one of the methods and systems herein described can be performed on one or more samples. In some of those embodiments, the methods can comprise

-   -   contacting the sample with an antibiotic to provide an         antibiotic treated sample, and     -   quantitatively detecting in the antibiotic treated sample a         nucleic acid of the microorganism, to obtain a detected         antibiotic treated nucleic acid concentration value of the         microorganism in the antibiotic treated sample.         wherein the quantitatively detecting is performed in presence of         a lysis treatment of the antibiotic treated sample targeting the         microorganism, the lysis treatment is performed following     -   pre-lysis mechanical separation of nucleic acid from the         microorganism in the antibiotic treated sample to obtain a         separated antibiotic treated sample comprising intracellular         nucleic acid of the microorganism, or     -   pre-lysis mechanical separation of the microorganism from the         nucleic acid in the antibiotic treated sample to obtain a         separated antibiotic sample comprising extracellular nucleic         acid of the microorganism.         In some embodiment, the method can be performed on a plurality         of tested samples to provide a plurality of antibiotic treated         samples, wherein in each antibiotic treated sample of the         plurality of antibiotic treated samples is provided by         independently contacting each antibiotic tested sample for a set         testing time, and in particular a testing time up to 120         minutes, and the quantitatively detecting is independently         performed following the contacting at a corresponding detection         time.

According to an eighth aspect, in any one of the methods and systems herein described the quantitatively detecting in the antibiotic treated sample a nucleic acid of the microorganism, can be performed by performing a non-lytic nucleic acid amplification and the nucleic acid concentration value can be provided in the form of time to positive.

According to a ninth aspect, in any one of the methods, systems herein described, the antibiotic treated sample is preferably provided by contacting the sample with an antibiotic and concurrently or after the contacting and before the detecting performing an enhancing treatment of the antibiotic treated sample with a non-ionic and/or a zwitterionic detergent. In some embodiments, the quantitatively detecting is performed in presence of a lysis treatment following pre-lysis separation of nucleic acid from the microorganism in the antibiotic treated sample, and the pre-lysis separation comprises contacting the sample with a nuclease to digest accessible nucleic acid thus separating the nucleic acid from the microorganism in the antibiotic treated sample.

According to a tenth aspect, any one of the methods, systems herein described can be performed on a plurality of antibiotic treated samples, wherein in each antibiotic treated sample of the antibiotic treated samples, the quantitatively detecting is independently performed

in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; or

in presence of a lysis treatment of the antibiotic treated sample targeting the microorganism, the lysis treatment following pre-lysis separation of nucleic acid from the microorganism in the antibiotic treated sample, or

in presence of a lysis treatment of the antibiotic treated sample targeting the microorganism, the lysis treatment following pre-lysis mechanical separation of the microorganism from the nucleic acid in the antibiotic treated sample,

to obtain a detected antibiotic treated nucleic acid concentration value of the microorganism in the antibiotic treated sample.

The antibiotic susceptibility test and related compositions, methods and systems herein described allow in several embodiments phenotypic measurements of antibiotic susceptibility and resistance of a microorganism (e.g. N gonorrhoeae).

The antibiotic susceptibility test and related compositions, methods and systems allow in several embodiments to perform an accurate and rapid antibiotic susceptibility test for microorganisms such as N. gonorrhoeae and carbapenem-resistant Enterobacteriaceae (CRE) based on quantification of DNA and/or RNA.

The antibiotic susceptibility test and related compositions, methods and systems herein described allow in several embodiments to provide an assay with a duration that shorter than the gold standard, through the short antibiotic exposure times and rapid nucleic acid quantification, enabling point of care timescales.

The antibiotic susceptibility test and related compositions, methods and systems herein described can be used in connection with various applications wherein identification and/or detection of antibiotic susceptibility for a microorganism is desired. For example, antibiotic susceptibility test and related compositions, methods and systems herein described can be used in drug research and to develop diagnostic and therapeutic approaches and tools to counteract infections, and to enable development and commercialization of narrow-spectrum antimicrobial therapeutics, such as antimicrobial therapeutics with a narrower spectrum than the therapeutic that would have been prescribed in the absence of the test. Additional exemplary applications include uses of the antibiotic susceptibility test and related compositions, methods and systems herein described in several fields including basic biology research, applied biology, bio-engineering, etiology, medical research, medical diagnostics, therapeutics, and in additional fields identifiable by a skilled person upon reading of the present disclosure.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and example sections, serve to explain the principles and implementations of the disclosure. Exemplary embodiments of the present disclosure will become more fully understood from the detailed description and the accompanying drawings, wherein:

FIG. 1 shows a schematic representation of an exemplary outcome of an exemplary antibiotic susceptibility test for a susceptible microorganism of the instant disclosure. In particular, Panel A shows a schematic representation of a control sample comprising the microorganism not treated with antibiotic and showing inaccessible DNA in an intact cell. Panel B shows a schematic representation of an antibiotic-treated sample showing a disrupted or lysed susceptible microorganism cell with DNA accessible to nuclease. Panel C shows a diagram illustrating the CT ratio of the control sample of Panel A and the antibiotic-treated sample of Panel B. A threshold control-treated (CT) ratio (the dashed line with a prediction of the cell being antibiotic susceptible (S).

FIG. 2 shows a schematic representation of an exemplary outcome of an exemplary antibiotic susceptibility test for a resistant microorganism of the instant disclosure. In particular, Panel A shows a schematic representation of a control sample comprising the microorganism not treated with antibiotic and showing inaccessible DNA in an intact cell. Panel B shows a schematic representation of an antibiotic-treated sample showing intact resistant microorganism cell. Panel C shows a diagram illustrating the CT ratio of the control sample of Panel A and the antibiotic-treated sample of Panel B. A threshold control-treated (CT) ratio (the dashed line with a prediction of the cell being antibiotic resistant (R).

FIG. 3 shows a diagram illustrating the results of experiments, described in Example 1, directed to detect the effects of antibiotic ceftriaxone (CRO) on DNA accessibility for ceftriaxone-susceptible Ng isolates (ng_3) and ceftriaxone-resistant Ng isolates (ng_30) in comparison to a control sample, with an increase in Cq corresponding to decrease in accessible DNA. Error bars represent 95% confidence intervals of the qPCR measurement.

FIG. 4 shows a diagram illustrating the results of experiments, described in Example 2, directed to detect effects of antibiotics CRO concentration on DNA accessibility for ceftriaxone-susceptible Ng isolates (ng_17) and ceftriaxone-resistant Ng isolate (ng_30). Error bars represent 95% confidence intervals. The diagram of FIG. 4 shows one untreated measurement of each isolate and two treated measurements.

FIG. 5 shows a diagram illustrating the results of experiments, described in Example 3, directed to detect time effect in a two-step incubation including a 30 or 50-minute incubation in a buffer for autolysis on DNA accessibility represented as Cq for S, a penicillin-susceptible Ng isolate (ng_17) and R, a penicillin-resistant Ng isolate (ng_44). Error bars represent 95% confidence intervals.

FIG. 6 shows a diagram illustrating the results of experiments, described in Example 3, directed to detect effect of an additional lysis agent Triton X in a two-step incubation including a 30 or 50-minute incubation in a TRIS buffer pH 8.5 on DNA accessibility represented as Cq for S, a penicillin-susceptible Ng isolate (ng_17), and R, a penicillin-resistant Ng isolate (ng_44).

FIGS. 7A-7C show diagrams illustrating the results of experiments, described in Example 4, directed to detect effect of β-lactams PEN (penicillin, FIG. 7A), CFM (cefixime, FIG. 7B), and CRO (ceftriaxone, FIG. 7C) on DNA accessibility represented as CT ratio and quantified with digital PCR. Error bars represent 95% confidence intervals

FIG. 8 shows a diagram illustrating the results of experiments, described in Example 5, directed to detect 16S rRNA concentration quantified with digital PCR, LAMP for antibiotic-susceptible and antibiotic-resistant Ng isolates in comparison to a control sample.

FIG. 9 shows a diagram illustrating the results of experiments, described in Example 5, directed to detect CT ratio for nucleic acid concentration, based on detection of 16S rRNA concentration quantified with digital PCR, LAMP for antibiotic-susceptible and antibiotic-resistant Ng isolates in comparison to a control sample as shown in FIG. 8.

FIG. 10 shows a diagram illustrating the results of experiments, described in Example 5, directed to detect effects of antibiotic ceftriaxone (CRO) on RNA accessibility by quantification of the RNA through RT-LAMP with Ng 16S rRNA primers for ceftriaxone-susceptible Ng isolates (ng_3) and ceftriaxone-resistant Ng isolates (ng_30) in comparison to control samples.

FIGS. 11A-11C show diagrams illustrating detection of nucleic acid concentrations following an AST performed according to exemplary methods of the disclosure, FIG. 11A shows that total nucleic acid concentration (NA=DNA+RNA) measured from the extraction FIG. 11B shows the total DNA concentration measured from the extraction. FIG. 11 C shows the C:T ratio computed from the total nucleic acid measurement (ddRT-PCR of extractions in the presence of a reverse-transcription enzyme), right. The concentrations are quantified in digital PCR.

FIGS. 12A and 12B shows diagrams illustrating detection of RNA concentration and calculations of the related CT ratio. In particular FIG. 12A shows a diagram illustrated the RNA concentrations computed from the subtraction of the DNA from the total NA as described in Example 6. FIG. 12B shows a diagram illustrating a CT ratio to differentiate between the susceptible and resistant isolates, giving a very similar readout to the CT ratio computed from the total RNA measurement. And conclusions made as with previous DNA accessibility CT ratios.

FIG. 13 shows a diagram illustrating the results of experiments described in Example 6, directed to detect the ratio of the measured DNA concentrations divided by the computed RNA concentration (total NA concentration minus DNA concentration) for ceftriaxone-susceptible Ng isolates (ng_3) and ceftriaxone-resistant Ng isolates (ng_30) in comparison to control samples.

FIG. 14 shows a schematic illustration of an exemplary AST according to the disclosure in which a penicillin-susceptible isolate is incubated with penicillin for 15 min-6 hours.

FIGS. 15A-15C show diagrams illustrating the results of an exemplary AST according to the disclosure in which beta-lactam treatment is performed in the absence of an enhancing treatment, as described in Example 7. Changes in nucleic acids between the untreated (FIG. 15A) and treated (FIG. 15B) shown as a CT ratio in FIG. 15C are a result of beta lactam exposure followed by a DNaseI degradation step to detect inaccessible nucleic acid.

FIG. 16 shows a schematic illustration of an exemplary AST according to the disclosure in which a penicillin-susceptible isolate is incubated with penicillin for 15 minutes, sonicated on the Covaris M220 sonicator (such as 30 s, 120 s), followed by a third incubation with DNaseI to degrade the accessible DNA as described in Example 8.

FIGS. 17A and 17B show diagrams illustrating the results of an exemplary AST according to the disclosure in which sonication is used an enhancement of the accessibility of DNA in a susceptible-treated sample compared to the susceptible-control sample. Experiments described in Example 8. FIG. 17A shows the raw qPCR measurements of N. gonorrhoeae 16S DNA with error bars of triplicate qPCR measurements. Sample ID of A1 corresponds to susceptible isolate ng_17 (untreated sample). Sample ID of B1 corresponds to susceptible isolate ng_17 treated with the antibiotic penicillin. Sample ID of A3 corresponds to the susceptible isolate ng_17 (untreated sample) with 30 s of covaris sonication at 15 W peak incident power. Sample ID of B3 corresponds to susceptible isolate ng_17 treated with the antibiotic penicillin with 30 s of covaris sonication at 15 W peak incident power. Sample ID of A5 is the sample as A3 but with 5 W peak incident power. Sample ID of B5 is the same as sample B3 but with 5 W peak incident power. FIG. 17B. shows the corresponding conversion to a CT ratio from each condition. The x-axis is sorted by the peak incident power of sonication, showing the relationship between changing this parameter and the efficacy of sonication as an enhancer. The power of 15 W for 30 s after antibiotic treatment enhances the ability of the DNaseI to degrade accessible DNAs and decrease the amount of inaccessible nucleic acids in the antibiotic-treated sample.

FIGS. 18A, 18B and 18C show diagrams illustrating the results of an exemplary AST according to the disclosure in which sonication is used an enhancement of the accessibility of DNA in a susceptible-treated sample compared to the susceptible-control sample. Experiments described in Example 9. FIG. 18A shows the untreated (A1, B1, C1) samples and treated samples (A3, B3, C3) for three conditions with a DNaseI treatment after sonication. Samples A1 and A3 did not undergo a sonication treatment after a 15-minute antibiotic exposure. Samples B1 and B3 underwent 30 seconds of sonication after the same 15-minute antibiotic exposure as A1 and A3. Samples C1 and C3 are the same as Samples B1 and B3 but with a 12-second of sonication. FIG. 18B shows the same samples as in FIG. 18A, but with an additional enhancer. Samples B2, B4, C2, and C4 all experienced two enhancers (1) sonication enhancer and (2) surfactant enhancer of 5 mM TNP for 5 minutes after sonication. FIG. 18C shows the conversion of these qPCR measurements into CT ratios. The open black circles represent the data from FIG. 18A, with DNaseI only and the black-filled-in circles represent data from FIG. 18B with the combination of both sonication and surfactant incubation.

FIG. 19 shows a schematic illustration of an exemplary AST according to the disclosure in which a ceftriaxone-susceptible isolate is incubated with ceftriaxone and DNaseI for 15 minutes on a Covaris M220 sonicator (with a protocol of 30 s sonication, 30 s holding at 37° C.). From there, there are two options. One, the DNaseI is inactivated at the remaining cells are lysed, followed by qPCR. Two, there is an additional 5-minute incubation in the presence of surfactant (10 mM CHAPS) and DNaseI to degrade the accessible DNA. The DNaseI will be inactivated simultaneously with the lysis of the remaining cells. In both cases, the quantified nucleic acids represent those from the cells with inaccessible DNA throughout the antibiotic exposure, soncation, and incubation with DNaseI as described in Example 9.

FIGS. 20A to 20C show diagrams illustrating the results of an exemplary AST according to the disclosure in which mechanical disruption by sonication is used as an enhancer for beta-lactam incubation, in combination with DNaseI as a degrader.

FIG. 21 shows a schematic illustration of an exemplary AST according to the disclosure in which a ceftriaxone-susceptible isolate is incubated with ceftriaxone and DNaseI for 15 minutes on a Covaris M220 sonicator (with a protocol of 30 s sonication, 30 s holding at 37° C.). From there, there are two options. One, the DNaseI is inactivated at the remaining cells are lysed, followed by qPCR. Two, there is an additional 5-minute incubation in the presence of surfactant (10 mM CHAPS) and DNaseI to degrade the accessible DNA. The DNaseI will be inactivated simultaneously with the lysis of the remaining cells. In both cases, the quantified nucleic acids represent those from the cells with inaccessible DNA throughout the antibiotic exposure, sonication, and incubation with DNaseI as described in Example 10.

FIGS. 22A-22B show a diagram illustrating the results of an exemplary AST according to the schematic in FIG. 21 performed without using the sample with subsequent surfactant incubation for the measurements. Experiments described in Example 10. These data plotted represent an isolate, ng_19 which is susceptible to ceftriaxone (CRO) and resistant to penicillin (PEN), and an isolate, ng_30, which is resistant to ceftriaxone (CRO). FIG. 22A illustrates the qPCR measurements, shown as Cqs with an error bar of a 95% confidence interval based on triplicate measurements. The “t0” measurements (the first bar of each pair) represent the initial amount of inaccessible nucleic acids in a sample. These samples have undergone a 5-minute DNaseI treatment, but no antibiotic exposure or enhancement treatments. The treated measurements are from the sonication enhancement and antibiotic incubation. FIG. 22B shows t0/TREATED ratio for isolates ng_19 treated with CRO, ng_30 treated with CRO, and ng_19 treated with PEN.

FIG. 23 shows a schematic illustration of an exemplary AST according to the disclosure in which an NG isolate is incubated with a beta-lactam antibiotic for 15 minutes, followed by a DNaseI digestion step in the presence of surfactant. At each time point, DNaseI will be added to degrade the accessible DNA, then the DNaseI will be inactivated simultaneously with the lysis of the remaining cells. The quantified nucleic acids represent those inaccessible from the DNaseI throughout the exposure protocol.

FIGS. 24A-24B show diagrams comparing the accessibility AST results for four isolates treated with 1 μg/mL ceftriaxone. Isolates ng_5, ng_17, and ng_19 are all susceptible to ceftriaxone, whereas, isolate ng_30 has reduced-susceptibility to ceftriaxone. The CT ratios can be used to differentiate between the antibiotic-susceptible (CT>1.5) and the antibiotic-resistant (CTR<1.5).

FIG. 25 shows LAMP amplification curves for four isolates as described in Example 12.

FIG. 26 shows individual LAMP amplification curves for each sample as described in Example 12.

FIG. 27 shows diagrams illustrating the average time-to-positive for LAMP amplification for each sample as described in Example 12.

FIG. 28 shows the average control (solid lines) and treated (dashed lines) PCR amplification curves for four isolates.

FIG. 29 shows the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates with RNase present.

FIG. 30 shows the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates with RT present.

FIG. 31 shows average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates as described in Example 13.

FIG. 32 shows diagrams illustrating the average time-to-positive for LAMP amplification as described in Example 13.

FIG. 33 shows triplicate control (solid lines) and triplicate treated (dashed lines) LAMP amplification curves for four isolates as described in Example 14.

FIG. 34 shows diagrams illustrating the percentage of DNA (top two plots) and RNA (bottom two plots) released after a 15 minutes incubation with ertapenem. The percentages were calculated relative to the total DNA and RNA concentration respectively.

FIG. 35 shows, for one E. coli strain, percentages of DNA detected by dPCR after filtration. The percentage was calculated relative to the total DNA concentration. The filtration was performed on four samples that underwent different exposure conditions, as described in Example 16.

FIG. 36 shows, for one E. coli strain, the percentage of DNA detected by dPCR after filtration. The percentage was calculated relative to the total DNA concentration. The filtration was performed on six samples that underwent different exposure conditions. The conditions involved contact or no contact with ertapenem, followed by one of sonication, heating, or contact with detergents. The conditions are described in Example 17.

FIG. 37 shows the average control (solid lines) and treated (dashed lines) LAMP amplification curves for two isolates as described in Example 18.

FIG. 38 shows diagrams illustrating the average time-to-positive for LAMP amplification as described in Example 18.

FIG. 39 shows the average control (black solid lines) and treated (black dashed lines) LAMP amplification curves for four isolates as described in Example 19.

FIG. 40 shows the average time-to-positive for LAMP amplification with samples extracted using the fully lysing condition as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold as described in Example 19.

FIG. 41 shows the average time-to-positive for each sample amplified directly in LAMP mix normalized to the fully lysing condition as described in Example 19.

FIG. 42 shows the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates as described in Example 20.

FIG. 43 shows diagrams illustrating the average time-to-positive for LAMP amplification as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold as described in Example 20.

FIGS. 44A-44B show diagrams illustrating the experimental results and data analysis methods as describe in Example 21. FIG. 44A shows the LAMP data for susceptible, labeled “S” on the x-axis and shown as open circles, and resistant, labeled “R” on the x-axis and shown as black circles isolates of E. coli exposed to four different antibiotics. The antibiotics include ampicillin (AMP), ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM). The y-axis shows the time-to-positive (TTP) measurement of the treated-sample adjusted for the TTP of the reference sample, which in this example is the DEB extracted nucleic acids quantified with a separate LAMP measurement. FIG. 44B shows the same measurements as FIG. 44A, but for a K. pneumoniae instead of E. coli. and with a different set of antibiotics. FIG. 44B shows K. pneumonie responses to the antibiotics ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM).

FIGS. 45A-45C show percentage change in DNA accessibility between the untreated and treated samples, at 15 min antibiotic exposure with PEN, CRO, and CFM as described in Example 22. All measurements were made in qPCR and the percent change was computed from the Cq difference in the untreated and treated samples. In each incubation a 15-minute antibiotic exposure is followed by an enhancing treatment for 5 minutes with 10 mM CHAPS. In the figure, all black circles represent measurements of antibiotic-susceptible isolates and open circles represent antibiotic-resistant isolates. In FIG. 45A N. gonorrhoeae isolates are tested with the antibiotic penicillin (PEN), with at least three biological replicates of each isolate. On the x-axis, a prefix of “S” refers to antibiotic-susceptible isolates and a prefix of “R” refers to an antibiotic-resistant isolate. In FIG. 45B N. gonorrhoeae isolates are tested with the antibiotic ceftriaxone (CRO), with at least three biological replicates of each isolate. On the x-axis, a prefix of “S” refers to antibiotic-susceptible isolates, a prefix of “E” refers to an isolate with an elevated MIC, and a prefix of “R” refers to an antibiotic-resistant isolate or an isolate with reduced-susceptibility to ceftriaxone. In FIG. 45C N. gonorrhoeae isolates are tested with the antibiotic cefixime (CFM), with at least three biological replicates of each isolate. On the x-axis, a prefix of “S” refers to antibiotic-susceptible isolates and a prefix of “R” refers to an antibiotic-resistant isolate.

FIGS. 46A-46C show percentage change in DNA accessibility at 15 min PEN treatment (FIG. 46 A), 30 min CRO treatment (FIG. 46 B) and 30 min CFM treatment (FIG. 46 C) for susceptible and resistant strains as described in Example 22. The plots are organized by the same layout and logic as FIG. 45, utilizing the same abbreviations for antibiotics and categorization of susceptible, elevated MIC, and resistant clinical isolates.

FIG. 47 illustrates a schematic representation of an exemplary embodiment of a polymerase-accessibility AST (pol-aAST) shown for susceptible and resistant samples exposed to β-lactams. In Panel (a): Treated aliquots are exposed to a β-lactam. In susceptible samples, β-lactams compromise cell wall integrity. In Panel (b): Nucleic acids (NAs) are released from compromised cells, increasing NA accessibility to polymerase. In Panel (c): Released NAs in the susceptible treated aliquot amplify faster than NAs from intact cells in the control aliquot, resulting in a difference in time-to-positive. No difference in amplification between control and treated aliquots is observed in resistant samples. In Panel (d): Time-to-positive difference (TTPD) between control and treated aliquots is used to assess susceptibility.

FIG. 48 shows an exemplary embodiment of pol-aAST using LAMP and PCR. Panel (a-b) show thermal profiles of LAMP and PCR. Panel (c-d) show LAMP and PCR amplification curves for a susceptible E. coli isolate exposed to ertapenem (ETP) for 15 min. Blue and black lines are the average of triplicate samples. Grey lines represent standard deviation of triplicates. A difference in time-to-positive (TTP) for control and treated aliquots is observed for susceptible isolates when quantifying nucleic acids using LAMP, but not PCR. Corresponding data are provided in S5 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 49 shows in some embodiments a percentage of DNA released following antibiotic exposure. Two susceptible (S) and two resistant (R) E. coli isolates were exposed to no antibiotic (control), ceftriaxone (CRO), ertapenem (ETP), or meropenem (MEM) for 15 min before filtering to separate intact cells from extracellular DNA. Experiments were performed in triplicate for all isolate/antibiotic combinations. Each point represents a single experiment; lines represent the average and standard deviation of replicate experiments. Corresponding data are provided in S6 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 50 shows in an exemplary embodiment a validation of the pol-aAST method using control (ctrl) and antibiotic-treated (+ABX) aliquots. Panel (a) illustrates a example calculation of time-to-positive difference (TTPD) between control and treated aliquots (TTPD_(CT)). The TTP (in min) of the control and treated aliquots are used to calculate TTPD_(CT). Panels (b-d) show graphs representing the pol-aAST results using Escherichia coli (b) Klebsiella pneumoniae (c), and Enterobacter spp. (d) isolates exposed to ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) for 15 min. Red points represent isolates with either no detectable carbapenemase genes (Ec and Kp isolates) according to a published genotypic assay[1] and commercial assay, [2] or no predictive genotype (Ebs isolates) according to the whole genome sequencing by the CDC.[3] S/R thresholds (dashed lines) were set halfway between the lowest susceptible (S) and the highest resistant (R) TTPD_(CT) values. Corresponding data are provided in S3 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 51 shows in an exemplary embodiment a validation of the pol-aAST method using lysed control (lc) and antibiotic-treated (+ABX) aliquots. Panel (a) illustrates an example calculation of time-to-positive difference (TTPD) between the lysed-control and antibiotic-treated aliquots (TTPD_(LT)). The TTP (in min) in the lysed-control and antibiotic-treated aliquots are used to calculate TTPD_(LT). Panels (b-d) show graphs representing the pol-aAST results using Escherichia coli (b) Klebsiella pneumoniae (c), and Enterobacter spp. (d) isolates exposed to ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) for 15 min. Red points represent isolates with either no detectable carbapenemase genes (Ec and Kp isolates) according to a published genotypic assay[1] and commercial assay, [2] or no predictive genotype (Ebs isolates) according to the CDC[3]. S/R thresholds (dashed lines) were set halfway between the lowest susceptible (S) and the highest resistant (R) TTPD_(LT) values except in the case of Enterobacter spp. treated with CRO (see text). Corresponding data are provided in S3 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 52 illustrates in an exemplary timed sample-to-answer pol-aAST using contrived urine samples spiked with either E. coli (Ec) or Klebsiella pneumoniae (Kp). In Panel (a): Because minimal sample handling is required for pol-aAST, all four contrived urine samples were run in parallel. In Panel (b): Urine samples were split into control and antibiotic-treated aliquots and incubated at 37° C. for 13 min. A timer was started immediately after sample splitting. In Panel (c): All samples were added to pre-made LAMP mix and run in technical triplicate. In Panel (d): Samples were amplified using LAMP and the fluorescence of reactions was monitored in real-time. Once total fluorescence passed a pre-determined threshold (indicating successful amplification), reactions were stopped and TTP values ported into an automated data-analysis spreadsheet. The timer was stopped as soon as the spreadsheet gave susceptibility calls. In Panel (e): Comparison of susceptibility calls with gold-standard AST categorization. Total assay time was 29.5 min. Corresponding data are provided in S3 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 53 shows a graph illustrating a pilot testing of pol-aAST with clinical UTI samples with a modified protocol. TTPD_(CT) values for ampicillin (AMP) and ertapenem (ETP) susceptibility obtained by pol-aAST, with clinical UTI samples containing E. coli. Each point represents the TTPD_(CT) value for one clinical sample tested once by pol-aAST (S2 and S4 Pola-Tables of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety). LAMP was performed in technical triplicate, see S4 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety, for values and statistical details.

FIG. 54 shows a graph illustrating the validation of pol-aAST TTPD_(CT) method using ampicillin. E. coli isolates were exposed to 16 μg/mL ampicillin (AMP) for 15 min. Threshold was set halfway between the lowest susceptible (S) and highest resistant (R) TTPD_(CT) value. Corresponding data are in S3 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 55 illustrates an exemplary schematic representation of a nuclease accessibility AST workflow performed on a sample containing a β-lactam susceptible pathogen. In Panel (a): A sample is split into control and treated aliquots; the treated aliquot is exposed to antibiotics (ABX) in the presence of DNase and any extracellular DNA is digested. ABX compromise peptidoglycan (PG) of cells in the treated aliquot. In Panel (b): Accessibility to nucleases is enhanced by the addition of an enhancer, which disrupts the outer membrane (OM). Genomic DNA becomes accessible and is degraded in the treated aliquot. Intact peptidoglycan in control samples (or in treated but resistant samples) prevents degradation. In Panel (c): Nucleic acids (NAs) are extracted, and DNase is inactivated. In Panel (d): Accessibility is quantified by measuring NA concentrations in the control and treated aliquots and dividing the amount of digested DNA by the amount in the control (to yield percentage accessibility). When the percentage accessibility is greater than the threshold (dashed line), the sample is categorized as susceptible.

FIG. 56 shows in a graph the percentage accessibility of DNA over time using the nuc-aAST without the addition of an enhancing step. Two penicillin-susceptible (PEN-S) and two penicillin-resistant (PEN-R) Ng isolates were exposed to penicillin in the presence of DNase I. DNA from the control and PEN-treated aliquots was extracted and quantified using qPCR at multiple time points to calculate percentage accessibility. Error bars represent the standard deviation of the PCR triplicates. Corresponding data are in S10 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 57 shows plots illustrating the results from a selection of exemplary enhancers. Six enhancers were tested for percentage of cell lysis due to enhancer alone (prior to antibiotic exposure) (Panels a-f); enhancement after 15-min exposure to penicillin (PEN) (Panels g-1); enhancement after 15-min exposure to ceftriaxone (CRO) (Panels m-r); and enhancement after 15-min exposure to cefixime (CFM) (Panels s-x). Each point represents a clinical isolate run as a single experiment for that condition. The shading indicates two preferred enhancers (TNP and CHAPS) All PCR was performed in technical triplicates with error bars representing the error in the PCR measurement propagated for the calculation of percentage lysis or percentage accessibility. All numerical values are available in S2 Nuca-Table and S3 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety. In particular, data for (Panels a-f) and (Panels g-x) are respectively reported in S2 Nuca-Table and S3 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 58 shows plots illustrating the validation of nuc-aAST using clinical isolates. Panels (a-c) show graphs representing nuc-aAST results after 15 min of exposure to (a) penicillin (PEN), (b) cefixime (CFM), and (c) ceftriaxone (CRO). Panels (d-e) show graphs representing nuc-aAST results after exposure to (d) CFM and (e) CRO for 30 min. Each point represents the average for a single isolate run in (at least) biological triplicate for that condition. All PCR was performed in technical triplicate. The dashed line represents the susceptibility threshold, which was set at 26.5% accessibility for 15-min exposures and 46% for 30-min exposures. Data plotted are reported in Nuca-Table S4; experimental data from individual replicates are detailed in S11 Nuca-Table and S12 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 59 illustrates a schematic workflow of a nuclease accessibility AST (nuc-aAST) with clinical urine samples and the accessibility results. In Panel (a) Clinical urine samples were concentrated by a factor of 5 and incubated with DNase I and saponin for 15 min to lyse host cells and clear free DNA. In Panel (b) The nuc-aAST protocol was performed in technical triplicates with no antibiotic (no ABX), ceftriaxone (CRO), or penicillin (PEN). Each nuc-aAST consisted of a 30-min exposure to 1 μg/mL CRO or PEN followed by a 3-5 min exposure to the enhancer TNP (see Methods for details). In Panel (c) Each clinical sample was isolated for gold-standard MIC testing and further experiments. In Panel (d) Results of six nuc-aASTs were performed directly on four clinical urine samples and susceptibility was determined (orange points). In parallel, isolates were prepared from these clinical samples and the results of the rapid nuc-aAST with clinical urine samples were compared to the same protocol with the prepared isolate (blue points). The means of the replicates are shown as horizontal lines (S7 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety). The minimum inhibitory concentration (MIC) was determined by the gold-standard method (51 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety). Error bars are the error in the PCR measurement propagated for the calculation of percentage lysis or percentage accessibility (S6 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety).

FIG. 60 illustrates in an exemplary embodiment the nuc-aAST workflow for contrived and clinical urine samples with each step timed. In Panel (a) The nuc-aAST workflow times for contrived samples are 15 min ABX exposure, 5 min enhancement, 2 min for DNA extraction, 8 min for dLAMP and AST call); timing for clinical urine samples include 30 min ABX exposure, 3-5 min enhancement, 10 min for extraction, and 20 min for dLAMP and AST call. In Panel (b) Amplification curves from which positive wells were determined and counted (a subset of 100 wells is shown for each microfluidic chip, (see S1-S4 Nuca-Data of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety). In Panel (c) A 2×2 mm subsection of masks was created from chips used for performing dLAMP on control and antibiotic-treated aliquots of susceptible and resistant samples; as an illustration, each mask shows ˜625 wells (out of ˜20,000 total wells) after 10 min of amplification. Wells that showed amplification of Ng DNA appear black. In Panel (d) The percentage of accessible DNA was determined at earliest significance (7-8 min of amplification; see Methods and S5 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety) for two penicillin-susceptible (S) and two penicillin-resistant (R) samples run using dLAMP (S5 Nuca-Table). Each step was timed individually and the sum-of-steps of the assay was 30 min. (e) Percentage accessibility was determined after 20 min of dLAMP for a representative technical replicate for each of the six nuc-aAST performed directly on four clinical urine samples. Ceftriaxone-susceptible and penicillin-intermediate samples are plotted together as not-resistant (NR) (S5 Nuca-Table; MICs are in 51 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety).

FIG. 61 shows in a graph an exemplary measurement of Cq after DNase is properly inactivated by extraction and heat treatment steps. After extraction/inactivation, Neisseria gonorrhoeae (Ng) DNA was spiked into the extractions containing the inactivated DNase I and incubated at 37° C. The Ng DNA was not degraded, confirming the inactivation of the DNase I enzyme. The concentration of DNase I, the composition of the incubations, and the extraction conditions were all performed under the same conditions as the ASTs. Error bars are 98% confidence intervals for three PCR replicates [4]. Corresponding, data are in S8 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 62 shows plots illustrating the qPCT results for the lambda DNA spike-in and the percentage of DNA digested calculated from qPCR results. In Panel (a) The qPCR results for the lambda DNA spike-in for three different urine samples. In Pancel (b) Percentage of DNA digested calculated from qPCR results. Corresponding data are in S9 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety.

FIG. 63 shows plots illustrating the susceptibility results from nuc-aAST using clinical isolates separated by minimum inhibitory concentration (MIC). Panels (a-c) Results of nuc-aAST after 15 min of exposure to (a) penicillin (PEN), (b) cefixime (CFM), or (c) ceftriaxone (CRO). (d-e) nuc-aAST results after exposure to (d) CFM or (e) CRO for 30 min. Each point represents the average for a single isolate run in (at least) biological triplicate for that condition; error bars represent the standard deviation from the biological replicates. All PCR assays were performed in technical triplicate. The dashed line represents the susceptibility threshold, which was set at 26.5% accessibility for 15-min exposures and 46% for 30-min exposures. Data are also plotted in FIG. 48. (Data plotted here are in Nuca-Table S4 of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety; experimental data from individual replicates are in S1l Nuca-Table and S12 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety; MICs are in 51 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety).

FIG. 64 shows plots illustrating Receiver operating characteristic (ROC) curves for different antibiotics. ROC curves generated from the data shown in FIG. 58, Corresponding data are reported in S4 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety. The false positive rate (FPR) is shown on the x-axis and the true positive rate (TPR) is shown on the y-axis. The area under the curve (AUC) is shown for each plot. Panels (a-c) show the results of the exemplary nuc-aAST after 15 min of exposure to (a) penicillin (PEN), (b) cefixime (CFM), or (c) ceftriaxone (CRO). Panels (d-e) The exemplary nuc-aAST results after exposure to (d) CFM or (e) CRO for 30 min.

FIG. 65 shows a chart reporting in the bottom part the results of ddPCR performed on filtrate of 20 samples (10 treated samples and 10 corresponding control samples) (see gray for treated and black for control dotted lines in the chart) and on total DNA extracted from 10 samples (solid line) following exposure to ertapenem of 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes

FIG. 66 shows a chart reporting variation of the percent extracellular nucleic acid concentration value calculated by comparing the extracellular concentration value of the filtrate and the total nucleic acid shown in FIG. 65 for the treated sample (light gray lines) and the control sample (dark gray line)

FIG. 67 shows a chart reporting in the bottom part the results of ddPCR performed on filtrate of 20 samples (10 treated samples and 10 corresponding reference treated samples) (see gray for treated and black for control dotted lines in the chart) and on total DNA extracted from 10 samples (solid line) following exposure to ertapenem of 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes

FIG. 68 shows a chart reporting variation of the percent extracellular nucleic acid concentration value calculated by comparing the extracellular concentration value of the filtrate and the total nucleic acid shown in FIG. 67 for the treated sample (light gray lines) and the reference sample (dark gray line)

FIG. 69 shows a chart reporting a version of the two graphs reported in FIG. 67 and FIG. 68 wherein instead of separate linear y axes, the two graphs are now plotted together on a single log₁₀ axis.

FIG. 70 shows a chart reporting in the bottom part the results of ddPCR performed on DNA (left panel) and DNA+RNA (right panel) from filtrate of 20 samples (10 treated samples and 10 corresponding reference treated samples) (see gray for treated and black for control dotted lines in the chart) and on total DNA extracted from 10 samples (solid line) following exposure to Ertapenem of 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes

FIG. 71 shows a chart reporting the results of ddPCR performed on genomic DNA (left panel) and ribosomal RNA (right panel) from filtrate of 20 samples (10 treated samples and 10 corresponding reference treated samples) (see black for treated and gray for control dotted lines in the chart) and on total DNA/RNA extracted from 20 samples (solid line) (10 treated samples and 10 corresponding reference treated samples) following exposure to ertapenem of 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes.

FIG. 72 shows a chart reporting the percent extracellular nucleic acid concentration value calculated by comparing the extracellular concentration value of the filtrate and the total nucleic acid shown in FIG. 71 for the reference sample (light gray lines) and the treated sample (black line)

FIG. 73 shows a chart reporting the results of ddPCR performed on genomic DNA (left panel) and ribosomal RNA (right panel) of Escherichia coli K12 from filtrate of 20 samples (10 treated samples and 10 corresponding reference treated samples) (see black for treated and gray for control dotted lines in the chart) and on total DNA/RNA extracted from 20 samples (solid line) (10 treated samples and 10 corresponding reference treated samples) following 30 minutes of exposure to ertapenem. The densities of the samples are chosen to have densities of 208000, 62500, 20800, 6250, 2080, 625, 208, and 0 cells/mL.

FIG. 74 shows a chart reporting the percent extracellular nucleic acid concentration value calculated by comparing the extracellular concentration value of the filtrate and the total nucleic acid shown in FIG. 73 for the reference sample (light gray lines) and the treated sample (black line).

FIG. 75 shows a chart reporting the results of ddPCR performed on genomic DNA (left panel) and ribosomal RNA (right panel) of E. coli clinical isolate from filtrate of 20 samples (10 treated samples and 10 corresponding reference treated samples) (see black for treated and gray for control dotted lines in the chart) and on total DNA/RNA extracted from 20 samples (solid line) (10 treated samples and 10 corresponding reference treated samples) following 30 minutes of exposure to ertapenem. The densities of the samples are chosen to have densities of 208000, 62500, 20800, 6250, 2080, 625, 208, and 0 cells/mL.

FIG. 76 shows a chart reporting the percent extracellular nucleic acid concentration value calculated by comparing the extracellular concentration value of the filtrate and the total nucleic acid shown in FIG. 75 for the reference sample (light gray lines) and the treated sample (black line).

FIG. 77 shows a graph of false positive rate as a function of APTV and three cell densities provided in connection with the experiments of Example 38.

DETAILED DESCRIPTION

Provided herein is an antibiotic susceptibility test for microorganisms and related compositions, methods and systems which is based on quantitative detection of nucleic acids of the microorganism following administration of the antibiotic to the microorganism.

The term “nucleic acid” or “polynucleotide” as used herein indicates an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group and that is the basic structural unit of nucleic acids. The term “nucleoside” refers to a compound (such as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. Accordingly, the term “polynucleotide” includes nucleic acids of any length, and in particular DNA, RNA, and fragments thereof. A polynucleotide of three or more nucleotides is also called “nucleotidic oligomer” or “oligonucleotide.”

The term “DNA” or Deoxyribonucleic acid” as used herein indicates a polynucleotide composed of deoxiribonucleotide bases or an analog thereof to form an organic polymer. The term “deoxyribonucleotide” refers to any compounds that consist of a deoxyribose (deoxyribonucleotide) sugar joined to a purine or pyrimidine base and to a phosphate group, and that are the basic structural units of a deoxyribonucleic acid, typically adenine (A), cytosine (C), guanine (G), and thymine (T). In an DNA adjacent ribose nucleotide bases are chemically attached to one another in a chain typically via phosphodiester bonds. The term “deoxyribonucleotide analog” refers to a deoxyribonucleotide in which one or more individual atoms have been replaced with a different atom with a different functional group. For example, deoxyribonucleotide analogues include chemically modified deoxyribonucleotides, such as methylation hydroxymethylation glycosylation and additional modifications identifiable by a skilled person.

The term “RNA” or “Ribonucleic acid” as used herein indicates a polynucleotide composed of ribonucleotide bases: or an analog thereof linked to form an organic polymer. The term “ribonucleotide” refers to any compounds that consist of a ribose (ribonucleotide) sugar joined to a purine or pyrimidine base and to a phosphate group, and that are the basic structural units of a ribonucleic acid, typically adenine (A), cytosine (C), guanine (G), and uracil (U). In an RNA adjacent ribose nucleotide bases are chemically attached to one another in a chain typically via phosphodiester bonds. The term “ribonucleotide analog” refers to a ribonucleotide in which one or more individual atoms have been replaced with a different atom with a different functional group. For example, ribonucleotide analogues include chemically modified ribonucleotides, such as methylation hydroxymethylation glycosylation and additional modifications identifiable by a skilled person. Examples of chemical modifications of RNA comprise dynamic modifications to RNA identified in the transcriptome, including N⁶-methyladenosine (m⁶A), inosine (I), 5-methylcytosine (m⁵C), pseudouridine (Ψ), 5-hydroxymethylcytosine (hm⁵C), and N¹-methyladenosine (m¹A), and related epitranscriptome which are described in Song and Yi 2017. [5] Additional chemical modifications of transfer RNA (tRNA) are described in Jackman and Alfonzo 2013 [6] Accordingly, the term RNA includes ribonucleic acids of any length including analogs or fragments thereof.

The term “antibiotic” sometimes abbreviated as ABX, as used herein refers to a type of antimicrobial used in the treatment and prevention of bacterial infection. Some antibiotics can either kill or inhibit the growth of bacteria. Others can be effective against fungi and protozoans. The term “antibiotic” can be used to refer to any substance used against microbes. Antibiotics are classified based on their mechanism of action, chemical structure, or spectrum of activity. Most antibiotics target bacterial functions or growth processes. Antibiotics having bactericidal activities target the bacterial cell wall, such as penicillins and cephalosporins, or target the cell membrane, such as polymyxins, or interfere with essential bacterial enzymes, such as rifamycins, lipiarmycins, fluoroquinolones, quinolones and sulfonamides. Antibiotics having bacteriostatic properties target protein synthesis, such as macrolides, lincosamides and tetracyclines. Antibiotics can be further categorized based on their target specificity. “Narrow-spectrum” antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria or a specific genus of bacteria. “Broad-spectrum” antibiotics affect a wide range of bacteria. Antibiotics can also be used in combinations with each other or with adjuvant substances (such as cilastatin or beta-lactamase inhibitors) that enhance their antimicrobial activity. These combinations are often approved by the Food and Drug Administration as distinct drug names.

In preferred embodiments, this method is used to analyze susceptibility and resistant antibiotics that directly or indirectly interact with cell envelope, structure and function, and integrity. e.g. by generating physiological stress, damaging essential cellular machinery, and causing cell death as will be understood by a skilled person. Exemplary antibiotics include beta-lactam antibiotics, and consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures. Including penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. Penams include narrow-spectrum penems such as, benzathine penicillin (benzathine & benzylpenicillin), benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), Procaine penicillin (procaine & benzylpenicillin), and Pheneticillin. Broad spectrum penams include amoxicillin and ampicillin. Extended spectrum penems include mecillinam, nafcillin, oxacillin, dicloxacillin, carboxypenicillins (including carbenicillin and ticarcillin), and ueidopenicillins (including azlocillin, mezlocillin, and piperacillin). Cephems include first, second, third, fourth, and fifth generation cephalosporins; including cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin, cefixime, cefdinir, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, and ceftaroline. Carbapenems include biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, and thienamycin. Monobactams include aztreonam, tigemonam, nocardicin A, and tabtoxinine b-lactam. Exemplary combinations of antibiotics and adjuvant substances include ampicillin/sulbactam, amoxicillin/clavulanate, ticarcillin/clavulanate, piperacillin/tazobactam, ceftazidime/avibactam, imipenem/cilastatin, and meropenem/vaborbactam. Other antibiotics that may impact the cell wall directly or indirectly include polymixin B, colistin, depolarizing antibiotics such as daptomycin, antibiotics that hydrolyze NAM-NAG, tyrothricin (Gramicidin or Tyrocidine), isoniazid, and teixobactin. Antibiotics that inhibit peptidoglycan chain elongation including vancomycin (Oritavancin Telavancin), teicoplanin (Dalbavancin), and ramoplanin. Antibiotics that inhibit peptidoglycan subunit synthesis and transport include NAM synthesis inhibition (fosfomycin), DADAL/AR inhibitors (Cycloserine), and bactoprenol inhibitors (bacitracin).

The wording “antibiotic susceptibility” or “antibiotic sensitivity” as used herein indicates the susceptibility of bacteria to antibiotics and the antibiotic susceptibility can vary within a species. Antibiotic susceptibility testing (AST) can be carried out to predict the clinical response to treatment and guide the selection of antibiotics as will be understood by a person skilled in the art. In some embodiments, AST categorizes organisms as susceptible, resistant, or intermediate to a certain antibiotic.

Microorganisms can be classified as susceptible (sensitive), intermediate or resistant based on breakpoint minimum inhibitory concentration (MIC) values that are arbitrarily defined and reflect the achievable levels of the antibiotic, the distribution of MICs for the organism and their correlation with clinical outcome. MIC value of a microorganism is the lowest concentration of an antibiotic that will inhibit its growth. Methods that can be used to measure the MIC of a microorganism comprise broth dilution, agar dilution and gradient diffusion (the ‘E test’), where twofold serial dilutions of antibiotic are incorporated into tubes of broth, agar plates or on a paper strip, respectively, as will be understood by a person skilled in the art. The disk diffusion method defines an organism as susceptible or resistant based on the extent of its growth around an antibiotic-containing disk. MIC values are influenced by several laboratory factors.

Laboratories follow standard for parameters such as incubation temperature, incubation environment, growth media, as well as inoculum and quality control parameters. In the U.S. Standards for performing AST as well as breakpoint MIC values for various bacteria can be found in Clinical & Laboratory Standards Institute (CLSI) publications, with an example also provided as Appendix A of U.S. Provisional Application No. 62/722,124 incorporated herein by reference in its entirety, as will be understood by the skilled person. In Europe, standards for performing AST as well as breakpoint MIC values for bacteria can be found in European Committee on Antimicrobial Susceptibility Testing (EUCAST) see http://www.eucast.org/clinical_breakpoints/at the time of filing of the instant disclosure) as will be understood by the skilled person.

The term “microorganism”, or “microbe” as used herein indicates a microscopic living organism, which may exist in its single-celled form or in a colony of cells, such as prokaryotes and in particular bacteria, and including fungi (yeast and molds), and protozoal parasites. Microorganisms include human and animal pathogens. Microorganisms can comprise one or more prokaryotes or individual genera or species of prokaryotes.

The term “prokaryotic” is used herein interchangeably with the terms “cell” and refers to a microbial species which contains no nucleus or other membrane-bound organelles in the cell. Exemplary prokaryotic cells include bacteria and archaea.

The term “bacteria” or “bacterial cell”, used herein interchangeably with the term “cell” indicates a large domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals, and are present in most habitats on Earth, such as terrestrial habitats like deserts, tundra, Arctic and Antarctic deserts, forests, savannah, chaparral, shrublands, grasslands, mountains, plains, caves, islands, and the soil, detritus, and sediments present in said terrestrial habitats; freshwater habitats such as streams, springs, rivers, lakes, ponds, ephemeral pools, marshes, salt marshes, bogs, peat bogs, underground rivers and lakes, geothermal hot springs, sub-glacial lakes, and wetlands; marine habitats such as ocean water, marine detritus and sediments, flotsam and insoluble particles, geothermal vents and reefs; man-made habitats such as sites of human habitation, human dwellings, man-made buildings and parts of human-made structures, plumbing systems, sewage systems, water towers, cooling towers, cooling systems, air-conditioning systems, water systems, farms, agricultural fields, ranchlands, livestock feedlots, hospitals, outpatient clinics, health-care facilities, operating rooms, hospital equipment, long-term care facilities, nursing homes, hospice care, clinical laboratories, research laboratories, waste, landfills, radioactive waste; and the deep portions of Earth's crust, as well as in symbiotic and parasitic relationships with plants, animals, fungi, algae, humans, livestock, and other macroscopic life forms. Bacteria in the sense of the disclosure refers to several prokaryotic microbial species which comprise Gram-negative bacteria, Gram-positive bacteria, Proteobacteria, Cyanobacteria, Spirochetes and related species, Planctomyces, Bacteroides, Flavobacteria, Chlamydia, Green sulfur bacteria, Green non-sulfur bacteria including anaerobic phototrophs, Radioresistant micrococci and related species, Thermotoga and Thermosipho thermophiles as would be understood by a skilled person. Taxonomic names of bacteria that have been accepted as valid by the International Committee of Systematic Bacteriology are published in the “Approved Lists of Bacterial Names” [7] as well as in issues of the International Journal of Systematic and Evolutionary Microbiology. More specifically, the wording “Gram positive bacteria” refers to cocci, nonsporulating rods and sporulating rods that stain positive on Gram stain, such as, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Cutibacterium (previously Propionibacterium), Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Nocardia, Staphylococcus, Streptococcus, Enterococcus, Peptostreptococcus, and Streptomyces. Bacteria in the sense of the disclosure refers also to the species within the genera Clostridium, Sarcina, Lachnospira, Peptostreptococcus, Peptoniphilus, Helcococcus, Eubacterium, Peptococcus, Acidaminococcus, Veillonella, Mycoplasma, Ureaplasma, Erysipelothrix, Holdemania, Bacillus, Amphibacillus, Exiguobacterium, Gracilibacillus, Halobacillus, Saccharococcus, Salibacillus, Virgibacillus, Planococcus, Kurthia, Caryophanon, Listeria, Brochothrix, Staphylococcus, Gemella, Macrococcus, Salinococcus, Sporolactobacillus, Marinococcus, Paenibacillus, Aneurinibacillus, Brevibacillus, Alicyclobacillus, Lactobacillus, Pediococus, Aerococcus, Abiotrophia, Dolosicoccus, Eremococcus, Facklamia, Globicatella, Ignavigranum, Carnobacterium, Alloiococcus, Dolosigranulum, Enterococcus, Melissococcus, Tetragenococcus, Vagococcus, Leuconostoc, Oenococcus, Weissella, Streptococcus, Lactococcus, Actinomyces, Arachnia, Actinobaculum, Arcanobacterium, Mobiluncus, Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Rothia, Stomatococcus, Brevibacterium, Cellulomonas, Oerskovia, Dermabacter, Brachybacterium, Dermatophilus, Dermacoccus, Kytococcus, Sanguibacter, Jonesia, Microbacteirum, Agrococcus, Agromyces, Aureobacterium, Cryobacterium, Corynebacterium, Dietzia, Gordonia, Skermania, Mycobacterium, Nocardia, Rhodococcus, Tsukamurella, Micromonospora, Propioniferax, Nocardioides, Streptomyces, Nocardiopsis, Thermomonospora, Actinomadura, Bifidobacterium, Gardnerella, Turicella, Chlamydia, Chlamydophila, Borrelia, Treponema, Serpulina, Leptospira, Bacteroides, Porphyromonas, Prevotella, Flavobacterium, Elizabethkingia, Bergeyella, Capnocytophaga, Chryseobacterium, Weeksella, Myroides, Tannerella, Sphingobacterium, Flexibacter, Fusobacterium, Streptobacillus, Wolbachia, Bradyrhizobium, Tropheryma, Megasphera, Anaeroglobus.

The term “proteobacteria” as used herein refers to a major phylum of Gram-negative bacteria. Many move about using flagella, but some are nonmotile or rely on bacterial gliding. As understood by skilled persons, taxonomic classification as proteobacteria is determined primarily in terms of ribosomal RNA (rRNA) sequences. The Proteobacteria are divided into six classes, referred to by the Greek letters alpha through epsilon and the Acidithiobacillia and Oligoflexia, including the alphaproteobacteria, betaproteobacteria and gammaproteobacteria as will be understood by a skilled person. Proteobacteria comprise the following genera: in the Alphaproteobacteria, Rickettsia, Ehrlichia, Anaplasma, Sphingomonas, Brevundimonas, Agrobacterium, Bartonella, Brucella, Ochrobactrum, Afipia, Methylobacterium, and Roseomonas; in the Betaproteobacteria, Burkholderia, Ralsonia, Alcaligenes, Achromobacter, Chromobacterium, Bordetella, Taylorella, Comamonas, Neisseria, Alysiella, Eikenella, Kingella, and Spirillum; in the Gammaproteobacteria, Xanthomonas, Stenotrophomonas, Cardiobacterium, Suttonella, Francisella, Legionella, Coxiella, Ricketsiella, Pseudomonas, Chryseomonas, Flavimonas, Oligella, Moraxella (Branhamella), Acinetobacter, Psychrobacter, Shewanella, Vibrio, Photobacterium, Aeromonas, Succinivibrio, Anaerobiospirillum, Ruminobacter, Succinimonas, Enterobacter, Brenneria, Budvicia, Buttiauxella, Calymmatobacterium, Cedeceae, Citrobacter, Edwardsiella, Erwinia, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pantoea, Plesiomonas, Proteus, Providencia, Rahnella, Salmonella, Serratia, Shigella, Tatumella, Trabulsiella, Yersinia, Yokenella, Pasteurella, Actinobacillus (Aggregatibacter), Haemophilus, and Mannheimia; in the Deltaproteobacteria, Desulfovibrio and Biophila; in the Epsilonproteobacteria, Campylobacter, Arcobacter, Helicobacter, and Wolinella. [8]. The Proteobacteria also comprise the species which are classified within the aforementioned genera. Within the Proteobacteria are the species Neisseria gonorrhoeae and Neisseria meningitidis within the class Betaproteobacteria, the order Neisseriales the family Neisseriaceae, and the genus Neisseria. It should be understood by the skilled practitioner that the classification and nomenclature of formal bacterial species is subject to revision as new scientific knowledge is discovered. Changes in name are performed according to rules in the International Code of Nomenclature of Bacteria, and future name changes can be found by consulting the International Journal of Systematic and Evolutionary Microbiology.

The term Enterobacteriaceae in the sense of the disclosure refers to members of the Proteobacteria that fall within the family Enterobacteriaceae, Class Gammaproteobacteria, as defined by the International Committee of Systematic Bacteriology. These bacteria are Gram-negative rods that can inhabit the gastrointestinal tracts of animals as well as environmental surfaces. Many species are pathogenic in humans and other animals. Many species are commensals that become pathogenic when their hosts immune barriers are breached. Enterobacteriaceae are frequently encountered in clinical specimens. [8] Enterobacteriaceae include the following taxa and clinical entities: Escherichia coli (E. coli), uropathogenic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, enteropathogenic E. coli, enteroinvasive E. coli, enterohemorrhagic E. coli, Shiga toxin-producing E. coli, diffusely adherent E. coli, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Citrobacter koseri (Citrobacter diversus), Salmonella enterica subsp. enterica and its serovars, Salmonella enterica Typhi, Salmonella enterica Paratyphi, Salmonella bongori, Shigella dysenteria, Shigella flexneri, Shigella boydii, Shigella sonnei, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Providencia stuartii, Edwardsiella hoshinae, Raoultella ornithinolytica, Raoultella planticola, Raoultella terrigena, Arizona hinshawii, Budvicia aquatica, Buttiauxella agrestis, Buttiauxella brennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae, Buttiauxella izardii, Buttiauxella noackiae, Buttiauxella warmboldiae, Cedecea davisae, Cedecea lapagei, Cedecea neteri, Cedecea species 3, Cedecea species 5, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmer, Citrobacter gillenii, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Edwardsiella ictaluri, Edwardsiella tarda, Edwardsiella tarda biogroup 1, Enterobacter amnigenus, Enterobacter asburiae, Enterobacter cancerogenus (Enterobacter taylorae), Enterobacter cowanii, Enterobacter dissolvens, Enterobacter gergoviae, Enterobacter hormaechei, Enterobacter intermedius, Enterobacter kobei, Enterobacter nimipressuralis, Enterobacter pyrinus, Enterobacter sakazakii, Erwinia spp., Ewingella americana, Hafnia alvei, Kluyvera ascorbate, Kluyvera cryocrescens, Kluyvera georgiana, Leclercia adecarboxylata, Leminorella grimontii, richardii, Moellerella wisconsensis, Morganella morganii, Obesumbacterium proteus, Pantoea agglomerans, Pantoea dispersa, Photorhabdus luminescens, Photorhabdus asymbiotica, Pragia fontium, Proteus hauseri, Proteus myxofaciens, Proteus penneri, Providencia alcalifaciens, Providencia heimbachae, Providencia rettgeri, Providencia rustigianii, Rahnella aquatilis, Serratia entomophilia, Serratia ficaria, “Serratia fonticola”, Serratia liquifaciens group, Serratia odorifera, Serratia plymuthica, Serratia rubidea, Tatumella ptyseos, Trabulsiella guamensis, Xenorhabdus nematophilus, Yersinia aldovae, Yersinia bercoviera, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, Yersinia rohdei, “Yersinia ruckeri”, Yokenella regensburgei.

The term carbapenem-resistant Enterobacteriaceae in the sense of this disclosure refers to any member of the family Enterobacteriaceae, defined earlier, that exhibit resistance to at least one member of the carbapenem class of antibiotics, defined earlier. The term carbapenem-resistant Enterobacteriaceae can be abbreviated as “CRE”. CRE isolates are frequently resistant to classes of beta-lactam antibiotics besides the carbapenems, namely the penicillins, cephalosporins, and monobactams. CRE isolates also frequently carry resistance toward other classes of antibiotics. Some CRE isolates are susceptible very few antibiotics, and some CRE isolates have been found to be resistant to all antibiotics available for use in humans in the USA or Europe. CRE achieve antibiotic resistance through a variety of resistance mechanisms, including the expression of enzymes that degrade beta-lactam antibiotics (carbapenemases, extended-spectrum beta-lactamases, and beta-lactamases), alterations in expression of their porin genes, and by unknown mechanisms. CRE prevalence has increased worldwide and in the USA in the past three decades. CRE cause a significant fraction of healthcare associated infections. CRE infections have an estimated 50% mortality rate in the USA.

In embodiments of the instant disclosure, methods and systems are described that are based on detection of a nucleic acid of a microorganism in a sample of an isolate or specimen treated with the antibiotic and in a sample of the isolate or specimen not treated with antibiotic.

The microorganism tested with methods and systems of the disclosure is generally referred in the present disclosure as target microorganism, and the nucleic acid detected in methods and systems of the instant disclosure is generally referred as target nucleic acid.

Accordingly, the term “target microorganism”, or “target microbe” as used herein indicates one or more microorganisms in the sample targeted specifically by the methods described herein. For example, when a sample contains a mixture of pathogens and commensal organisms, the target microorganism of methods and system herein described can be one or more of the pathogens in the mixture.

The term “target nucleic acid” as used herein indicates a nucleic acid of a target microorganism detected by methods described herein. In particular in the nucleic acid detection methods described herein, one or more nucleic acids from the target organism may be targeted for detection.

The term “sample” as used herein indicates a limited quantity of something that is indicative of a larger quantity of that something, including but not limited to fluids from an isolate or a specimen such as biological environment, cultures, tissues, commercial recombinant proteins, synthetic compounds or portions thereof. In particular biological samples can comprise one or more cells of any biological lineage, as being representative of the total population of similar cells in the sampled individual. Exemplary biological samples comprise the following: whole venous and arterial blood, blood plasma, blood serum, dried blood spots, cerebrospinal fluid, lumbar punctures, nasal secretions, sinus washings, tears, corneal scrapings, saliva, sputum or expectorate, bronchoscopy secretions, transtracheal aspirate, endotracheal aspirations, bronchoalveolar lavage, vomit, endoscopic biopsies, colonoscopic biopsies, bile, vaginal fluids and secretions, endometrial fluids and secretions, urethral fluids and secretions, mucosal secretions, synovial fluid, ascitic fluid, peritoneal washes, tympanic membrane aspirate, urine, clean-catch midstream urine, catheterized urine, suprapubic aspirate, kidney stones, prostatic secretions, feces, mucus, pus, wound draining, skin scrapings, skin snips and skin biopsies, hair, nail clippings, cheek tissue, bone marrow biopsy, solid organ biopsies, surgical specimens, solid organ tissue, cadavers, or tumor cells, among others identifiable by a skilled person. Biological samples can be obtained using sterile techniques or non-sterile techniques, as appropriate for the sample type, as identifiable by persons skilled in the art. Some biological samples can be obtained by contacting a swab with a surface on a human body and removing some material from said surface, examples include throat swab, nasal swab, nasopharyngeal swab, oropharyngeal swab, cheek or buccal swab, urethral swab, vaginal swab, cervical swab, genital swab, anal swab, rectal swab, conjunctival swab, skin swab, and any wound swab. Depending on the type of biological sample and the intended analysis, biological samples can be used freshly for sample preparation and analysis, or can be fixed using fixative. Preferably, in methods and systems herein described, the sample contains live target microorganisms. In methods and systems herein described a sample can be split in two or more parts (also indicated as sub-samples, aliquots or partitions) each including a smaller quantity of the original sample, and thus providing a sample of the original sample, as will be understood by a skilled person.

The term “isolate” as used herein indicates a portion of matter resulting from a separation of a strain of a microorganism from a natural, usually mixed population of living microbes, as present in a natural or experimental environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora.

The word “specimen” as used herein indicates a portion of matter from an environment for use in testing, examination, or study. The environment can comprise individuals and in particular human beings. In these instances, a specimen can include a portion of tissues, organs or other biological material from the living being such as urethra, urine, cervix, vagina, rectum, oropharynges, conjunctiva, or any body fluids.

When referred to as a noun, the term “individual” as used herein in the context of treatment refers to a single biological organism, including but not limited to, animals and in particular higher animals and in particular vertebrates such as mammals and in particular human beings.

In particular, in embodiments of the instant disclosure, methods and systems are described that are based on detection of a differential concentration of nucleic acid of a target microorganism in a sample of an isolate or specimen treated with the antibiotic and in a sample of the isolate or specimen not treated with antibiotic.

In embodiments herein described, a differential concentration of a nucleic acid can be expressed with fold change approach. In the fold-change approach, a nucleic acid is considered to have a differential concentration if the ratio of a nucleic acid concentration value, possibly normalized, between two nucleic acids exceeds a certain threshold. The two nucleic acid concentration value can be obtained from a sample under different conditions (e.g. antibiotic treated and untreated conditions) or at different times under a same or different condition (e.g. on a same treated sample at different times or on a treated sample and an untreated sample at different times).

For a sample wherein detection of a nucleic acid concentration value is performed according to a set method, a nucleic acid concentration value is a value obtained by quantitively detecting a target nucleic acid in the sample within the set method. A nucleic acid concentration value in the sense of the disclosure is a value proportional to the true concentration of the target nucleic acid in the sample Any positive number can be used as the proportionality constant, preferably the proportionality constant equals to 1.

In some embodiments, the nucleic acid concentration value is a true concentration that can be measured in units of copies per volume (also indicated as number concentration, number density), moles per volume (also indicated as molarity or molar concentration), mass of nucleic acid per volume (also indicated as mass concentration), copies per mass of solvent, moles per mass of solvent (also indicated as molality), mass of nucleic acids per mass of solvent also indicated as mass fraction), volume fraction, normality, and any other mathematical conventions for expressing chemical concentrations. In these embodiments, the nucleic acid concentration value can be detected by a digital quantification method such as digital PCR (dPCR).

In some embodiments, the nucleic acid concentration value is not the true concentration but proportionally reflects the amount of nucleic acid in the sample. That is, for a higher amount of true nucleic acid concentration in the sample, a higher nucleic acid concentration value will be obtained. In those embodiment, proportionality can refer to a linear proportionality, or to any strictly monotonic function of the nucleic acid concentration as will be understood by a skilled person.

In some of these embodiments, the nucleic acid concentration value can be a direct measurement from experiments. For example, the nucleic acid concentration can be estimated by detecting a nucleic acid with a digital quantification method such as digital PCR (dPCR), or with correction for amplification efficiency by digital LAMP or digital RPA or other digital isothermal amplification chemistries, or calculated from the number of reads corresponding to the target nucleic acids as measured by many high throughput sequencing methods.

Alternatively, digital methods and other methods could be used to provide a concentration parameter that is proportional to concentration, such as raw concentration or positive counts obtained from digital LAMP or digital RPA or other digital isothermal amplification chemistries, or from sequencing such as from the number of reads corresponding to the target nucleic acids as measured by many high throughput sequencing methods. In some of the digital methods, correction for Poisson loading of nucleic acid molecules is used to obtain the concentration parameter from the raw data, as would be known to those skilled in the art.

In other embodiments, the nucleic acid concentration value can be obtained by detecting a concentration parameter such as Cq and time-to-positive (reaction time), fluorescence intensity, UV/visible light absorbance, and comparing the detected concentration parameter with a standard calibration curve to obtain the nucleic acid concentration value. The Cq and the time-to-positive are examples of reaction times, since those parameters measure how long it takes for a given nucleic acid amplification reaction to create a certain amount of amplified product. In particular, the Cq, also known as the quantification cycle, threshold cycle (Ct) or take-off-point (TOP), is the PCR cycle number at which a reaction crosses a threshold value. The time-to-positive (TTP) is a parameter indicating the time it takes for a non-cycling isothermal amplification reaction to create an amount of product above a threshold value. Accordingly, the TTP of a reaction is analogous to the Cq of PCR reactions, the difference being that the units of the Cq are PCR cycle numbers, while the units of TTP are units of time such as seconds or minutes as will be understood by a skilled person.

in one exemplary embodiment, a nucleic acid concentration value can be obtained from a detected Cq value by using the formula “nucleic acid concentration value”=2{circumflex over ( )}(−Cq) or if a more specific determination is desired=n{circumflex over ( )}(−Cq), where n is a real number called the “PCR per cycle efficiency” that represents the effect number of new nucleic acids created from one nucleic acid over the span of one PCR cycle. Accordingly, the n value can be values such as 1.5 to 1.9, as will be understood by a skilled person. The PCR per cycle efficiency is found by 1) calculating a summary statistic (such as the arithmetic mean, the geometric mean, the harmonic mean, the median, the mode, or any other summary statistic existing in the literature, or any other combination of the above summary statistics) on the ratios of PCR readouts (e.g. fluorescence readouts) of each pair of adjacent PCR cycles during real-time quantitative PCR; 2) performing ordinary least squares regression, linear regression, non-linear regression, or other statistical techniques identifiable by a skilled person in connection with real-time quantitative PCR readouts or mathematical transformations of the real-time quantitative PCR readouts; or 3) any methods for calculating the PCR per cycle efficiency according to methods identifiable by a skilled person.

In another exemplary embodiment, a nucleic acid concentration value can be obtained from a concentration parameter such as detected reaction time in terms of a time-to-positive (TTP) of an exponential quantification method, such as an isothermal amplification method, by using the formula “nucleic acid concentration value”=n{circumflex over ( )}(−reaction time) wherein the reaction time is provided as TTP where n has typically a value larger than 1, and reflects the properties of the detecting reaction. For example, if the isothermal exponential amplification doubles the concentration of the product nucleic acid every 20 seconds, then the relative concentration=2{circumflex over ( )}(−reaction time in terms of TTP in seconds/20 seconds). In this example, if the isothermal amplification causes a 6-fold increase in the nucleic acid every 10 seconds, then the relative concentration=6{circumflex over ( )}(−TTP in seconds/10 seconds). For a reaction where nucleic acid amplification is considered linear and not exponential, such as rolling circle amplification, then n is equal to 1, there is an inverse linear dependence of reaction time on starting target nucleic acid concentration, and the “nucleic acid concentration value”=1/(reaction time in terms of TTP). (see Example 2-30 and 43).

In yet another exemplary embodiment, a nucleic acid concentration value can be obtained from a detected florescence by using the formula relative concentration=n*(fluorescence intensity) where n is a normalization factor determined by constructing a standard calibration curve.

In yet another exemplary embodiment, a nucleic acid concentration value can be obtained from a detected UV light or visible light absorbance by using the Beer-Lambert Law. The Beer-Lambert law states that A=εlc, where A is the absorbance, ε is the molar attenuation coefficient or absorptivity (known in the literature for DNA and RNA), l is the length of the light path (determined by spectrophotometer design), and c is the molar concentration that is being measured. UV/vis spectrophotometers measure UV/visible light absorbance at many wavelengths and are commonly used to calculate nucleic acid concentrations as will be understood by a skilled person.

When two samples derived from the same sample are measured by the same method, the proportionality constant connecting nucleic acid concentration value and true concentration is approximately the same and therefore it does not need to be known to calculate the nucleic acid concentration ratio.

In methods herein described, detection of a differential concentration of nucleic acid is performed by detecting a concentration parameter of the nucleic acid in samples of isolates or specimens with and without antibiotic treatment and then obtaining a nucleic acid concentration value for the treated and a nucleic acid concentration value for a reference sample such untreated samples respectively. In some embodiments, when the detection technique is a digital quantification method, the nucleic acid concentration value is the detected value of the parameter.

The terms “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. The “detect” or “detection” as used herein can comprise determination of chemical and/or biological properties of the target, including but not limited to ability to interact, and in particular bind, other compounds, ability to activate another compound and additional properties identifiable by a skilled person upon reading of the present disclosure. The detection can be quantitative or qualitative. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A quantitative detection in the sense of the disclosure comprises detection performed semi-quantitatively, above/below a certain amount of nucleic acid molecules as will be understood by a skilled person and/or using semiquantitative real time isothermal amplification methods including real time loop-mediated isothermal amplification (LAMP) (see e.g. semi quantitative real-time PCR). For a given detection method and a given nucleic acid input, the output of quantitative or semiquantitative detection method that can be used to calculate a nucleic acid concentration value or nucleic acid concentration ratio (NACR) is a “concentration parameter”.

In methods herein described where the target nucleic acid comprises DNA and/or RNA, quantitative detection of nucleic acid concentration can be performed with various techniques (commonly in combination with reverse transcription for RNA) such as by nucleic acid sequencing, including RNA-seq, DNA-seq, Sanger sequencing, next-generation sequencing, Nanostring, and other pore-based sequencing technologies; polymerase chain reaction (PCR) including quantitative real-time PCR (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), and multiplexed versions of such PCR techniques; and isothermal techniques such as loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase reaction (RPA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA) for whole genome amplification (WGA), nicking enzyme amplification reaction (NEAR), and additional reactions identifiable by a skilled person, and digital versions of these isothermal amplifications; hybridization such as nucleic acid microarrays and fluorescence in situ hybridization; optical measurements such as absorbance measurements such as UV/vis spectroscopy and fluoroscopy (e.g. quantification of nucleic acids by a nucleic-acid specific fluorescent dye); electrochemical measurements of nucleic acid concentration, such as those that measure redox reaction currents enabled by binding of nucleic acids to a functionalized electrode. Additional techniques include Nanostring as well as high throughput DNA and RNA sequencing as reads per kilobase per million reads (RPKM) or transcripts per million (TPM) for RNA-seq data and additional nucleic acid quantification techniques identifiable to a skilled person. It should be understood that in such methods quantitative detection of expression of a gene is commonly combined with a reverse transcription step to convert the RNA sequence into a cDNA sequence which can be quantified by methods described herein and/or identifiable by a skilled person. Either sequence-specific or sequence-non-specific primers can be used to initiate reverse transcription of a target gene as will be understood by a skilled person.

In some embodiments where the target nucleic acid comprises RNA, detecting nucleic acid concentrations can be performed at the transcription level by performing RNA-seq and calculating RNA concentration values based on the sequence data.

In some embodiments where the target nucleic acid comprises RNA, the RNA concentration values can be detected and provided as transcripts per million (TPM) as will be understood by a person skilled in the art. In particular, to calculate TPM, read counts are first divided by the length of each gene in kilobases, which gives reads per kilobase (RPK). RPKs for all genes are added and the sum is divided by 1,000,000. This gives the “per million” scaling factor. Finally, the RPK value for each genes is divided by the “per million” scaling factor to give TPM. [9]

In particular, in embodiments herein described, method and systems herein described, quantitatively detecting the nucleic acid concentration is performed in treated samples of the isolate or specimen following treatment of the samples with the antibiotic and in reference samples of the isolate or specimen without treatment with the antibiotic.

“Reference samples” as used herein often indicates samples providing a standard for comparison against an antibiotic treated sample where the factor being tested (here antibiotic treatment) is applied during a testing procedure. Reference samples are used to produce reference nucleic acids.

A part of the treated sample can be used as a reference sample, obtained for example by splitting and processing the treated sample. A second sample treated under the same or different conditions as the first treated sample may also be used as a reference sample.

Reference samples can be control samples, which are samples subjected to the same testing procedure as another corresponding sample, except that the factor being tested is not applied. Reference samples and treated samples can be derived by splitting and manipulating the original sample being tested by the methods herein described. (see e.g. Example 38 to 42)

Total target nucleic acid refers to total intracellular and extracellular nucleic acid from the target microorganisms in a sample. In some embodiments, when gene-specific detection methods are used, such as nucleic acid amplification methods, total target nucleic acid refers to the total detectable target nucleic acid by the method used. It may include DNA or the sum of DNA and RNA amounts for some quantification methods used, such as RT-PCR. A reference sample may be different from a treated sample in the way it is handled prior to detection of nucleic acids, such as in the incubation conditions, or it might differ from a treated sample in the detection methods (such as a different target molecule in the same extraction). A reference sample may provide the total target nucleic acid in a sample (no treatment, no degradation) by undergoing complete lysis through a lysis treatment (as in Example 21 and Example 38 to 42).

A reference sample may be a quantification of the total inaccessible nucleic acid target molecules in a sample (no treatment with antibiotic, but includes a degrader or a separation step to remove the accessible nucleic acids), (as in Example 10). A reference sample may be nucleic acid molecules in a treated sample, but a molecule which is not targeted by a degrader (see Example 6; RNase is included to degrade accessible target RNA molecules but not target DNA molecules; therefore, DNA can be an appropriate reference sample that comes from the same sample as the treated sample). (see e.g. Example 38 to 42).

In some of these embodiments, providing a treated sample and a reference sample of the isolate or specimen can comprise contacting a first sample of the isolate or specimen with a treatment media to obtain a control sample and contacting a second sample of the isolate or specimen from the same source or host with the same treatment media and an antibiotic to obtain a treated sample. The contacting time (referring to the duration of the contact) with the treatment media can differ from the treated sample and the reference sample by more than 30%. Additionally, the treatment medias can differ between the reference and the treated samples by one or more components, such as the presence or absence of degrading molecules such as nucleases. Some embodiments include a lysis treatment of the reference sample but not necessarily in the treated sample, such as in Example 21 and 38 to 42. A method which uses three or more sample measurements for a ratio includes any scenario normalized to another normalization nucleic acid. For example, control samples and treated samples can be used to generate a CT ratio, which can then be normalized to a ratio of starting nucleic acid concentrations in the respective control and treated samples. This can be used to correct for technical or biological variability, such as uneven division of a sample containing the microorganism before antibiotic exposure, by normalizing to a third (or reference) condition.

Some embodiments include a lysis treatment of a reference sample to obtain total nucleic acid but not necessarily in the treated sample, such as in Example 21. For example, treated samples can be compared to the total nucleic acid concentrations in the respective control sample. This can be used to correct for technical variability, such as uneven division of a sample containing the microorganism before antibiotic exposure (see also Examples 38 to 42).

In some particular treatment of a sample with a treatment media is performed to create a controlled environment that would minimize the impact of biochemical parameters of a sample, such as pH or salt concentration or presence of molecules other than RNA or cells (human cells or other microorganisms other than target microorganism) on the RNA and DNA and cell-envelope response of the target microorganism to an external stimulus such as an antibiotic treatment and/or quantitative detection of gene expression. Treatment media can be used to create a more controlled environment for obtaining a more reliable response and/or gene expression. For example, treatment media can be composed of commercially available broths designed for the cultivation of microorganisms (such as Fastidious Broth or Cation-Adjusted Mueller-Hinton Broth from Hardy Diagnostics) or prepared using chemically defined components. In some cases, commercial broths can be diluted to create the desired treatment environment. For example, a specific osmolarity (for example in the range 0.0-0.5 osmols) or pH (for example in the range 5-9). Treatment media can be modified to contain specific factors to increase or decrease the metabolism of the target microorganism (such as carbon source or specific anions or cations). Gentle or vigorous mixing can be performed at specific time intervals after the addition of microorganisms to the treatment media in order to maintain homogeneity and reliable gene expression.

In some embodiments, a control sample and/or treated sample of the isolate or specimen or sample can preferably be pretreated to enrich said sample with nucleic acids or with the target microorganism, and/or to remove human nucleic acid or nucleic of other microorganisms and/or other organisms. The removal of human nucleic acid can be performed via hybridization to beads or columns with probes specific for human nucleic acid. The removal of human nucleic acid can also be performed via selective lysis of human cells and degradation of released human nucleic acid. The sample may also be pretreated to enrich or deplete, as desired, nucleic acid via size selection. The removal of human DNA can be performed with treatment with DNases.

In embodiments, treatment or exposure with antibiotic can be performed by adding antibiotics to the microorganism and incubating the sample under certain condition preferably following and/or upon contacting the sample with a treatment media.

Treatment media used in connection with antibiotic exposure in accordance to methods herein described, can be designed to support physiological processes of the target microorganism, enable or accelerate remodeling or modification or disruption of the cell envelope, and promote interaction of the microorganism and antibiotic. Accordingly, the treatment media can be selected to include a source of energy and nourishment specific for the target microorganism, such as providing elements such as carbon, hydrogen, oxygen, nitrogen phosphorus, Sulphur, potassium, magnesium, calcium, iron, trace elements and organic growth factors which can be provided as organic sources such as simple sugars e.g. glucose, acetate or pyruvate, amino acids, nitrogenous bases or extracts such as peptone, tryptone, yeast extract and additional identifiable by a skilled person. Inorganic sources such as; carbon dioxide (CO₂) or hydrogen carbonate salts (HCO₃)NH₄Cl, (NH₄)₂SO₄, KNO₃, and for dinitrogen fixers N₂, KH₂PO₄, Na₂HPO₄, Na₂SO₄, H₂S, KCl, K₂HPO₄, MgCl₂, MgSO₄, CaCl₂, Ca(HCO₃)₂, NaCl, FeCl₃, Fe(NH₄)(SO₄)₂, Fe-chelates1), CoCl₂, ZnCl₂, Na₂MoO₄, CuCl₂, MnSO₄, NiCl₂, Na₂SeO₄, Na₂WO₄, Na₂VO₄, as well as vitamins, amino acids, purines, pyrimidines (see the website https://www.sigmaaldrich.com/technic al-documents/articles/microbiology/microbiology-introduction.html at the filing date of the present disclosure). Additional parameters considered to select the proper treatment media for a target microorganism comprise osmotic pressure, pH, oxygen content, water content, carbon dioxide content as will be understood by a skilled person to support physiological processes of the target microorganism, enable or accelerate DNA replication and translation, maintain cellular uniformity and homogeneity in suspension, and promote interaction of the microorganism and antibiotic. For example in some of the experiments described herein with reference to N. gonorrhoeae the treatment media used was Fastidious Broth from Hardy Diagnostics (cat no. K31) which comprise pancreatic Digest of Casein, Yeast Extract, Dextrose, Peptic Digest of Animal Tissue, Sodium Chloride, Brain Heart Infusion, TRIS, Pancreatic Digest of Gelatin, Agarose, L-Cysteine HCl, Magnesium Sulfate, Ferrous Sulfate, Hematin, NAD, Pyridoxal and Tween® 80 (see https://catalog.hardydiagnostics.com/cp_prod/content/hugo/fbbroth.htm at the filing date of the present disclosure) Additional treatment media suitable to support physiological processes of N. gonorrhoeae, E. coli, K. pneumoniae, or other target microorganisms to enable or accelerate DNA replication and translation, maintain cellular uniformity and homogeneity in suspension, and promote interaction of the microorganism and the antibiotic are identifiable by a skilled person (see also Example 23).

In methods herein described, incubation of a sample with an antibiotic can be performed at a temperature such that a physiological response to the antibiotic is generated in the target microorganism. Incubation temperature is within ±0.5 degrees, ±1 degree, ±2 degrees, ±3 degrees Celsius from the physiological (for the organism) conditions. For example, for many human pathogens the physiological T is 37 C and the antibiotic incubation will be carried out at 37 degrees Celsius±0.5 degrees, ±1 degree, ±2 degrees, ±3 degrees Celsius). Also, adding the antibiotics can be performed throughout incubation or at set intervals during incubation to increase or decrease the physiological response of the microorganism to the antibiotic. In some embodiments, temperatures varying from 37 degrees Celsius might be used as a viability preserving enhancer or enhancer as an enhancing treatment alone, or with another enhancer. Temperature as a viability preserving enhancing treatment creates stress but preserves viability according to the definition of the viability preserving enhancing treatment provided herein. More generally, enhancing treatments that are more than 5 degrees, more than 10 degrees, more than 15 degrees, more than 20 degrees, more than 25 degrees, more than 30 degrees Celsius, can be used as enhancing treatments. For example, a viability preserving treatment for E. coli can be up to 75 degrees Celsius.

In some embodiments, concentrations of antibiotics above the resistance breakpoint are used, see Example 2. In particular in some embodiments, the antibiotic for treating the sample herein described can be provided at a concentration equal to or above the breakpoint MIC for the susceptible isolate or specimen to the antibiotic. In particular, the antibiotic for treating the sample herein described can be provided at a concentration lower than the breakpoint MIC for the resistant isolate or specimen to the antibiotic, for example 1.5 times (or 1.5×) lower, 2 times (or 2×) lower, 3 times (or 3×) lower, 4 times (or 4×) lower, 8 times (or 8×) lower, or 16 times (or 16×) lower than the breakpoint MIC for a resistant isolate. In some embodiments, the antibiotic for treating the sample herein described is provided at a concentration higher than the breakpoint MIC for the resistant isolate or specimen to the antibiotic, for example 1.5 times (or 1.5×) higher, 2 times (or 2×) higher, 3 times (or 3×) higher, or 4 times (or 4×) higher, 8 times higher (8×), 16 times higher (or 16×) than then breakpoint MIC. The breakpoint MIC of the antibiotic can be obtained from the Clinical & Laboratory Standards Institute (CLSI) guidelines, European Committee of Antimicrobial Susceptibility Testing (EUCAST) or other sources identifiable to a skilled person. In some embodiments, samples can be treated at several concentrations of the antibiotics for example, to measure the MIC of an organism before identifying the marker of antibiotic susceptibility as will be understood by a skilled person. One antibiotic susceptibility test can include exposing one or more aliquots of the sample to more than one antibiotic concentration.

These concentrations include multiple dilutions below the susceptible MIC breakpoint, dilutions between the susceptible and resistant MIC breakpoints (including intermediate breakpoint concentrations), as well as a dilution above the resistant MIC breakpoint. To determine, degree of susceptibility, the sample can be exposed to three concentrations of antibiotic: a concentration equal to the susceptible MIC breakpoint, a concentration equal to the concentration of the resistant MIC breakpoint, and a concentration equal to the average of the maximum and minimum of the intermediate MIC breakpoint range. Susceptibility can then be determined, for example, by measuring the slope obtained by fitting a curve or line to the three points on the T:R ratio vs treatment concentration plot, and/or by comparing the relative difference in T:R ratio between the low and intermediate concentration of antibiotic and the difference in T:R ratio between the intermediate and high concentration, and/or by comparing the magnitude of the value relative to a pre-defined threshold, or a combination of these analyses.

In some embodiments of the methods of the instant disclosure, the time period of contacting the sample with an antibiotic can be up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, 25 minutes, 30 minutes, up to 45 minutes, up to 60 up to 90 up to 120 up to 360 or higher, inclusive of any value therebetween or fraction thereof.

In some embodiments of the methods of the instant disclosure, the time period of contacting the sample with an antibiotic is shorter than the doubling time of the target organism. For example, the time of contacting could be less than 1× doubling time, less than 0.75× doubling time, less than 0.5 doubling time, less than 0.35 doubling time, less than 0.25 doubling time, less than 0.2 doubling time, less than 0.15 doubling time, less than 0.1 doubling time, less than 0.075 doubling time, less than 0.05 doubling time. In one example of this disclosure, example 7, antibiotic exposure times greater than the doubling time, or many doubling times can be used, but in the absence of growth-sustaining media for the target microorganism where substantial (greater than or equal to one doubling of the initial detected target nucleic acid) replication of DNA is not observed in the untreated sample.

In embodiments herein described, the detection of a nucleic acid specific for the microorganism is performed either

-   -   in absence of a lysis treatment of the antibiotic treated sample         targeting the target microorganism; or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         performed following pre-lysis separation of nucleic acid from         the microorganism in the antibiotic treated sample, or     -   in presence of a lysis treatment of the antibiotic treated         sample targeting the microorganism, the lysis treatment         performed following pre-lysis separation of microorganism from         the nucleic acid in the antibiotic treated sample,         to obtain a detected antibiotic treated nucleic acid         concentration value of the microorganism in the antibiotic         treated sample.

The wording “lysis,” “lyse,” and “lysing” as used herein indicates disruption of the cell membranes and release of intracellular contents which results in death of the cell. As will be understood by a skilled person, cell death can be measured by measuring of cell death or cell viability according to one or more measurement methods such as serial dilution on plate to quantify CFU/mL [13] most probable number (MPN) assays [14], LIVE/DEAD flow cytometry (such as kits available through ThermoFisher scientific), Live/Dead viability staining assays cytometry (such as kits available through ThermoFisher scientific), and automated cell counters (such as the QUANTOM Tx Microbial Cell Counter from Logos Biosystems), metabolic assays and metabolic stains and additional methods identifiable by a skilled person.

A skilled person will understand that that cells of different organisms can undergo lysis under different conditions, and that lysis conditions for mammalian cells can be different that lysis conditions of the microorganism cells. Accordingly, a treatment directed to lyse one or more cell in a sample can be set up based on the type of cells targeted (e.g. bacterial or mammalian) and the composition of the reference mixture as well as reaction conditions such as pH temperature and osmolarity of the reaction mixture.

Lysis in the sense of the disclosure can occur by mechanisms including natural cell death, as well lytic agents produced by cells or added exogenously, or environmental stresses.

A “lytic agent” in the sense of the disclosure indicates any substance or energy that that results in lysis of a target cell if applied to the target.

Lytic agents in the sense of the disclosure comprise chemical lytic agent such as detergents and/or enzyme capable of catalyzing disassembly of cell walls, mechanical methods capable of disrupting the cell wall or membrane such as sonication at Covaris M220 sonication parameters 75 W peak incident power, >15% duty cycle, >200 cycles per burst, and >30 minutes in a volume of 50 uL such as high pH or high temperature (see Examples 3). Examples of chemical lytic agents suitable to perform a lysis of the disclosure Triton X-100, Tween-20, SDS, NP-40, and Lysozyme. Examples of mechanical lytic agents suitable to perform a lysis of the disclosure include sonication.

Lytic agents in the sense of the disclosure can be used to perform a lysis treatment of a sample and/or an enhancing treatment of a sample.

A “lysis treatment” in the sense of the disclosure is a concurrent combined or sequential administration of lytic agents that results in lysis of ≥90%, preferably ≥95%, more preferably ≥97%, and even more preferable ≥99% target cells in a control sample. Depending on the target organism, a lysis treatment can be obtained by exposing the organisms to high and low extremes incubation condition, which will depend on the type and features of the target cells. Exemplary lysis treatment in the sense of the disclosure for some target microorganism comprises high pH, such as pHs greater than 11 for 30 minutes or more, high temperatures such as >90 C for 10 minutes or more. A skilled person will be able to identify the correct conditions for a lysis treatment depending on the taxonomy of the target cell. For example, lytic treatment of Gram-positive cell can be performed with additional enzymatic treatment of the cell wall in combination or in parallel with the above listed conditions. Lytic treatment of a gram negative like N. gonorrhoeae can be performed by any one of the conditions above.

Accordingly, lysis treatment of target microorganism in the sense of the disclosure can be performed using lytic agents at conditions directed to result in the lysis of ≥90% or microorganism in the sample. For example, the ionic detergents such as SDS or BAC at concentrations above their critical micelle concentrations (CMC) and/or sonication at powers greater than (Covaris M220 sonication parameters 75 W peak incident power, >15% duty cycle, >200 cycles per burst, and >30 minutes in a volume of 50 uL) for gram negative organism and at higher powers such as 5×, 10×, 100× the power used for gram-negative organisms. Examples of conditional lytic agents suitable to perform a lysis treatment of the disclosure include pHs greater than 8 (see Examples 3) and temperatures greater than 90 C for >1 min.

In some embodiments, lysis treatment of target microorganism in the sense of the disclosure can be performed, for example, with a commercial lysis kits such as that provided by Zymo or Qiagen. For gram-negative microorganisms, such kit can include highly denaturing lysis agents containing guanidinium salts in combination with buffers and enzymes to promote complete disruption of all cell envelope and denaturation of cellular proteins.

A “stressor” is a reagent of a form of energy that acts synergistically with antibiotic to disrupt cell envelope.

An “enhancement treatment” or an “enhancing treatment” in the sense of the disclosure refers to a concurrent combined or sequential administration of lytic agents and or stressors that results in in lysis of <90%, preferably ≤60%, more preferably ≤30%, and even more preferable ≤35%, more preferably ≤15% most preferably ≤5% target cells in a control sample. An enhancing treatment used together with the antibiotic exposure in the antibiotic treated sample is directed to preserve the viability of at least 10% of microorganism in a sample.

Exemplary enhancing treatment in the sense of the disclosure for most target microorganism comprises pH above optimal physiological conditions for the cell, such as pHs greater or equal to 7.5 and lower than 9, or equal or less than 6.5 and greater than 5 for 30 minutes or less, high temperatures such as >38 C and <80 C for 30 minutes or less depending on the temperature selected, or high or low osmolarity values deviating from the physiological osmolarity by up to 250 mOsmole for 30 minutes or less, in some embodiments applied in a form of osmotic shock, in some embodiments approaching zero osmolarity. A skilled person will be able to identify the correct conditions for a lysis treatment depending on the taxonomy of the target cell. For example, lytic treatment of Gram-positive cells can be performed with additional enzymatic treatment of the cell wall in combination or in parallel with the above listed conditions. Lytic treatment of a Gram-negative like N. gonorrhoeae can be performed by any one of the conditions above.

In some embodiments herein described, an enhancer increases nucleic acid accessibility to nuclease in cells that have a compromised cell wall as a result of antibiotic exposure, while minimizing lysis of cells and imposing minimal or no effect on downstream extraction and quantification of nucleic acids. In some embodiments, the enhancers used herein results in a percentage of cell lysis lower than 90%, preferably lower than 50%, and even more preferably lower than 25%.

In some preferred embodiments, an enhancer used for the enhancement treatment herein described comprises one or more non-ionic or zwitterionic detergent. (see e.g. Examples 32 to 37).

Non-ionic detergents are characterized by their uncharged, hydrophilic headgroups. Examples of non-ionic detergents include Tween, Triton, the Brij series and others identifiable to a person skilled in the art.

Zwitterionic detergents are a type of detergents having polar head groups containing both negatively and positively charged atomic groups, resulting in the overall charge of their polar head groups being neutral. Examples of zwitterionic detergents include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), CHAPS 0 (also known as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), myristyl sulfobetaine (also known as. SB3-14, 3-(N,N-dimethylmyristylammonio)propanesulfonate), octyl sulfobetaine (also known as 3-(N,N-dimethyloctylammonio)propanesulfonate), ZWITTERGENT 3-08 Detergent (also known as .n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), other sulfobetaine compounds, and others identifiable to a person skilled in the art (see e.g. https://www.gbiosciences.com/Zwitterionic-Detergents and https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14572925 at the filing date of the present disclosure).

A lysis treatment in the sense of the disclosure typically results in conversion of ≥90%, ≥95%, ≥97%, ≥99% of the total intracellular nucleic acids of the target cell to extracellular nucleic acids of the target cell.

Thus in embodiments of the disclosure where the method comprises a lysis treatment and/or an enhancing treatment results in an increase in accessible nucleic acid of the target cells and in a decrease in the inaccessible nucleic acid of the sample as will be understood by a skilled person upon reading of the disclosure.

The wording “accessibility” in the sense of the disclosure refers to the capability of a nucleic acid comprised in a referenced mixture, to react with a referenced reagent in the referenced mixture (typically a sample) under referenced conditions (the conditions of a steps or assay of a method).

The wording “accessibility” in the sense of the disclosure refers to the capability of a nucleic acid comprised in a referenced mixture, to react with a referenced reagent in the referenced mixture (typically a sample) under referenced conditions (the conditions of a steps or assay of a method).

Accordingly, “an accessible nucleic acid” in the sense of the disclosure is a nucleic acid capable of reacting with a referenced reagent capable within a referenced mixture under referenced conditions. Conversely an “inaccessible nucleic acid” in the sense of the disclosure is a nucleic acid incapable of reacting with a referenced reagent within a referenced mixture under referenced conditions.

Reagents added in samples or other referenced mixtures of methods of the instant disclosure typically include degrading reagents (including exonucleases and endonucleases, RNases, DNases, restriction enzymes, and other enzymes that cleave or degrade nucleic acids), detecting reagents (including enzymes used in detection of nucleic acids, including reverse transcriptases, polymerases, primers, probes), or binding reagents (including primers, probes, affinity reagents, nucleic acid binding proteins, surfaces for binding nucleic acids such as silica surfaces). Reacting includes binding as will be understood by a skilled person.

In methods herein described a key factor impacting accessibility is cell permeability of the nucleic acid and the reagent under the conditions of a certain step or assay performed in accordance with the method.

Cell permeability in the sense of the disclosure indicates the collection of mechanisms that regulate the passage of a referenced solute and in particular nucleic acid through biological membranes and/or a cell envelope as will be understood by a skilled person depending on the cell at issue.

A cell membrane indicates lipid bilayers that can contain proteins embedded. A cell envelope indicates the inner cell membrane and the cell wall of a bacterium. In gram-negative bacteria the cell envelope can comprise an outer membrane. Bacterial cell envelopes fall into two major categories: a Gram-positive type and a Gram-negative type, distinguished by Gram staining as will be understood by a skilled person.

Accordingly, in methods herein described cell permeability and refers to the ability of the majority of a nucleic acid or an externally added reagent to pass through the envelope of target cells in the sample. For example, a reagent can be considered cell impermeable when intracellular concentration of the added reagent is less than 5% of the extracellular concentration, less than 1% of the extracellular concentration, less than 0.1% of extracellular concentration, less than 0.01% of the extracellular concentration. Typically, intracellular nucleic acids are not accessible to cell-impermeable reagents. Typically, extracellular nucleic acids are accessible to cell-impermeable reagents.

Lysis of cells performed in accordance with a lysis treatment and/or an enhancing treatment in the sense of the disclosure affect the target nucleic acid accessibility in the methods of the disclosure as will be understood by a skilled person upon reading of the present disclosure.

In some embodiments of the methods herein described the detection of nucleic acid of a target microorganism is performed in absence of a lysis treatment in the sense of the disclosure or in presence of a lysis treatment of the antibiotic treated sample following pre-lysis separation of microorganism from the nucleic acid in the antibiotic treated sample. In those embodiments, the method is directed to detect a nucleic acid of the target microorganism that is accessible to detection reagents in the sample following the contacting and before the detecting.

In some embodiments of the methods herein described detection of nucleic acid of a target microorganism is performed in presence of a lysis treatment in the sense of the disclosure of the antibiotic treated sample following pre-lysis separation of nucleic acid from the target microorganism in the antibiotic treated sample. In those embodiments the method is directed to detect a nucleic acid of the target microorganism that is inaccessible to detection reagents in the sample following the contacting and before the lysing treatment.

In some embodiments of all the methods herein described wherein the method comprises performing an enhancing treatment of the sample concurrently or after contacting the sample with the antibiotics.

In some embodiments herein described directed to detect accessible or inaccessible nucleic acid, the method can comprise isolating the nucleic acid from the antibiotic treated sample before detecting.

In those embodiments, in methods wherein detection of nucleic acid of a target microorganism is performed in absence of a lysis treatment in the sense of the disclosure, the isolating is performed by purifying the nucleic acid from the sample in absence of lysis in the sense of the present disclosure. Exemplary methods for isolating nucleic acids without lysis include isothermal amplification techniques such as qLAMP; methods that separate cells from liquid by non-lysing mechanical forces or particle size such as filtration by membranes, sieves, or microfluidic devices, centrifugation, sedimentation, flocculation, gel electrophoresis, electrophoresis, isotachophoresis, dielectrophoresis, and size-exclusion chromatography; methods that separate cells from liquid by chemical affinity for bacterial cells such as affinity chromatography; and other approaches identifiable by a skilled person upon reading of the present disclosure. (see e.g. Examples 25-30)

In embodiment methods wherein detection of nucleic acid of a target microorganism is performed in presence of a lysis treatment in the sense of the disclosure of the antibiotic treated sample following pre-lysis separation, (either directed to remove the microorganism from the nucleic acid in the sample to detect accessible nucleic acid, or to remove nucleic acid from the microorganism in the sample to detect inaccessible nucleic acid) the isolating is performed by extracting the nucleic acid from the sample with procedure which may comprise lysis in the sense of the disclosure. Exemplary nucleic acid isolating methods include combinations of techniques that lyse—such as alkaline lysis with SDS [13], bead beating, centrifugation, French presses, Dounce homogenizers, rotor homogenizers, ultrasonic homogenizers, and pressure homogenizers—and techniques which separate nucleic acids out of a liquid phase, such as phenol-chloroform extraction, incubation with nucleases, precipitation and concentration by alcohols, gel electrophoresis followed by gel dissolution; affinity chromatography; commercial nucleic acid extraction kits (e.g. Qiagen Miniprep Kit, Lucigen DNA Extraction Buffer); and other approaches identifiable by a skilled person upon reading of the present disclosure, (see e.g. Examples 32 to 37 and Examples 38 to 42).

In embodiments herein described directed to detect accessible or inaccessible nucleic acid, the method can comprise separation to remove either the accessible nucleic acid or the inaccessible nucleic acid from the sample.

The wording “separate” or “separation” as used herein indicates an action performed on a sample such that two desired components of the sample (such as nucleic acid and cells of a target microorganism) are no longer able to come into molecular contact. One example of such separation include filtration through a filter with a pore size (such as 0.2 um) such that cells are removed from surrounding liquid and any components of the surrounding liquid smaller than the pore size of the filter. One example of such separation includes selective degradation of a selected component (such as degradation of extracellular nucleic acids (NAs) with DNase I) such that the component is removed from the sample.

In some embodiments of methods directed to detect accessible nucleic acid and comprising a separation, the separation is directed to remove the microorganism (intracellular nucleic acid of the microorganism) from the nucleic acid in the sample to provide a separated sample comprising the extracellular nucleic acid of the microorganism. The separation can be performed mechanically e.g. by filtering the sample to remove the retentate from the sample formed by the filtrate, or by centrifugation followed by selective removal of the pellet from the supernatant, sedimentation followed by selective removal of the sediment, chromatography followed by selection of the target fraction. Other approaches identifiable by a skilled person upon reading this disclosure may be used to separate the microorganism from nucleic acids in the sample.

In some embodiments of methods directed to detect inaccessible nucleic acid, the separation is directed to remove the nucleic acid (extracellular nucleic acid of the microorganism) from the microorganism in the sample to provide a separated sample comprising the intracellular nucleic acid of the microorganism. The separation can be performed mechanically e.g. by filtrating the sample to remove the filtrate from the sample formed by the retentate, or by centrifugation followed by selective removal of the supernatant from the pellet, sedimentation followed by selective removal of the supernatant, chromatography followed by selective of the target fraction. Other approaches identifiable by a skilled person upon reading this disclosure may be used to separate the nucleic acids from microorganisms in the sample.

In some embodiments wherein methods and systems herein described comprise mechanical separation, the mechanical separation can be preferably performed by filtration, centrifugation or more preferably by filtration under vacuum and/or in combination with centrifugation or other techniques to increase the flow of the sample through the filter as will be understood by a skilled person. In some of those embodiments filtration can be performed with filters of a solid, gelatinous, granular, woven, or other porous material containing pores no larger than a desired size; the just-mentioned filtration with or without a subsequent wash of a liquid substance; filtration using porous substances that specifically and reversibly bind bacteria using covalent or non-covalent chemical bonds, or other separation techniques identifiable by a skilled the skilled person (see also Examples 38-42). Filter membranes comprising filter membrane of any polymeric material that does not dissolve or react with the intended liquid sample. Filter membrane materials preferably do not exhibit high binding to any intended analyte chemical species, but if binding is detected, coating with a blocking agent mitigates the loss of analyte. Example blocking agents include salmon sperm DNA, yeast tRNA, any nucleic acid not derived from the target microorganisms, bovine serum albumin, and milk powder. Example filter membrane materials compatible with the aqueous solutions used in the disclosed same sample filtration AST include cellulose nitrate, cellulose acetate, regenerated cellulose, mixed cellulose ester, nitrocellulose, nylon, polyethersulfone (i.e. PES, polysulfone), polytetrafluoroethylene (i.e. PTFE, Teflon™), polyvinylidene fluoride, polycarbonate, glass fibers, borosilicate glass fibers, quartz fibers, paper, and hardened paper. If the filter membrane is of a material not wettable by the intended liquid sample, the membrane may be coated by detergents. If detergents interfere with downstream applications, they can be removed with a wash step in which the intended liquid (e.g. water) is passed through the filter shortly before use.

In some embodiments of methods directed to detect inaccessible nucleic acid, when the separation is directed to remove the nucleic acid from the microorganism in the sample the separation can be performed chemically by performing a nuclease treatment of the sample.

In those embodiments of methods directed to detect inaccessible nucleic acid, the method can further comprise adding nucleases, such as DNase (Examples 1-4) or RNase (Examples 5-6) to the antibiotic treated sample. In some embodiments, for example when beta lactam antibiotics are used, antibiotic susceptibility is indicated by increased accessibility of the pathogen's nucleic acids to the nuclease in the treated sample relative to the control sample.

In some of those embodiments of methods directed to detect inaccessible nucleic acid, nucleases can be added during the ABX exposure step (Examples 1-2, 4-6). In some of those embodiments, nucleases can be added during the exposure to lytic agents (Examples 1-6).

In methods herein described, to during the incubation, the sample can be collected at different time interval for further analysis. In addition to collecting samples during the incubation with antibiotics, samples can be collected for analysis before treatment or exposure. Such samples can be used as controls in analysis. Detection of response of the target microorganism to the antibiotic can be performed one or more times at any time after antibiotic treatment or exposure. In some embodiments, rapid detection, for example detection completed within 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes during or after exposure.

In some of embodiments providing a treated sample and a control sample of the isolate or specimen can comprise enriching a first sample and a second sample of the isolate or specimen from the same source or host with the microorganism to obtain control, or reference, samples respectively, and contacting the second sample with an antibiotic to obtain an antibiotic treated sample respectively.

In embodiments of the method and systems herein described, providing a treated sample and a control sample of the isolate or specimen can comprise enriching a first sample and a second sample of the isolate or specimen from the same source or host with the microorganism, contacting the first sample with a treatment media following the enriching to obtain the control samples respectively and contacting the second sample of the isolate or specimen from the same source or host with the same treatment media and an antibiotic to obtain an antibiotic treated sample respectively. The sample may be split into two or more parts or sub-samples, such as a scenario wherein many antibiotic treatments are compared to one control, or reference, sample. Multiple treated samples can be setup and sampled at different times, or, a treated sample can be split in sub-samples during treatment and analyzed.

In methods herein described enriching a sample with the target microorganisms can be performed between sample collection (and optionally elution from a collection tool such as a swab) and exposure. In particular enriching a sample with target microorganisms and in particular bacteria (such as Neisseria gonorrhoeae or Escherichia coli) can be performed by capturing the target microorganism using a solid support (e.g. a membrane, a filtration membrane, an affinity membrane, an affinity column) or a suspension of a solid reagent (e.g. microspheres, beads). Capture of a target microorganism can improve the assay and the response to antibiotic. Capture can be used to enrich/concentrate low-concentration samples. Capture followed by washing can be used to remove inhibitors or components that may interfere with the method described here. Capture followed by washing may be used to remove inhibitors of nucleic acid amplification or inhibitors of other quantitative detection assays. Enrichment can also be performed using lysis-filtration techniques to lyse host cells and dissolve protein and/or salt precipitates while maintaining bacterial cell integrity then capturing target bacteria on filters (e.g. mixed cellulose ester membranes, polypropylene and polysulfone membranes, and more particularly cellulose nitrate, cellulose acetate, regenerated cellulose, mixed cellulose ester, paper, nitrocellulose, nylon, polyethersulfone—also known as. PES, polysulfone) polytetrafluoroethylene also known as PTFE, Teflon™, and polyvinylidene fluoride as will be understood by a skilled person). Enrichment can also be performed by binding target bacteria to membranes of microspheres, optionally coated with an affinity reagent (e.g. an antibody, an aptamer) specific to the target bacteria's cell envelope. When microspheres or beads are used for capture, they can be filtered, centrifuged, or collected using a magnet to enrich bacteria. AST in the format described here can then be performed directly on captured bacteria, or the bacteria can be released before performing the method.

In some embodiments, the methods herein described further comprise detecting a nucleic acid concentration ratio in the sample by comparing the detected antibiotic treated nucleic acid concentration value with a detected reference nucleic acid concentration value of the nucleic acid of the target microorganism in the sample.

A reference nucleic acid concentration value as used herein indicates a target nucleic acid concentration value obtained from a reference sample. Reference nucleic acids may be obtained from a reference sample, which differs from a treated sample in one or more of the following: handling prior to detection of nucleic acids (such as in the incubation conditions, or the presence or absence of lysis treatments) or it might differ from a treated sample in the detection methods (such as a different target molecule in the same extraction). A reference nucleic acid may be the total target nucleic acid in a sample (no treatment, degradation) which has undergone complete lysis through a lysis treatment (as in Example 21). A reference nucleic acid may be a quantification of the total inaccessible nucleic acid target molecules in a sample (no treatment with antibiotic, but includes a degrader or a separation step to remove the accessible nucleic acids), (as in Example 10). A reference nucleic acid may be nucleic acid in a treated sample, but a molecule which is not targeted by a degrader (see Example 6; RNase is included to degrade accessible target RNA molecules but not target DNA molecules; therefore, DNA can be an appropriate reference nucleic acid that comes from the same extraction as the treated nucleic acid).

A reference sample can have target nucleic acids quantified and compared to the target nucleic acids treated sample to generate a T:R ratio (also may be referred to as T:R) to assess susceptibility compared to a threshold of T:R ratio under those conditions. (see e.g. Examples 38 to 42).

In some embodiments the reference nucleic acid concentration value is a control nucleic acid concentration value obtained in a control sample of the isolate or specimen comprising the target microorganism not treated with the antibiotic. (see e.g. Examples 38 to 42).

In particular, in embodiments of the methods performed in absence of a lysis treatment of the antibiotic treated sample, the control acid concentration value can be obtained following detection in the control sample performed in absence of a lysis treatment of the control sample.

In embodiments of the methods performed in presence of a lysis treatment of the antibiotic treated sample, the control acid concentration value can be obtained following detection in the control sample performed in presence of a lysis treatment of the control sample. In particular, in some of these embodiments wherein the lysis treatment is performed following pre-lysis separation of nucleic acid from the microorganism in the antibiotic treated sample, the detection of nucleic acid in the control sample is also performed following pre-lysis separation of the nucleic acid from the microorganism in the control treated sample.

In those embodiments instead wherein the lysis treatment is performed following pre-lysis separation of microorganism from the nucleic acid in the antibiotic treated sample, the detection of nucleic acid in the control sample is also performed following pre-lysis separation of the microorganism from the nucleic acid in the control treated sample as will be understood by a skilled person.

Accordingly, in all embodiments comprises methods herein described, quantitative detection of a nucleic acid concentration can be performed to provide a control nucleic acid concentration value C in a control sample not treated with the antibiotic and a corresponding nucleic acid treated concentration value T in a treated sample treated with the antibiotic for a specimen or isolate.

In particular, in embodiments of the methods herein described wherein a reference (or control) nucleic acid concentration is performed, quantitative detection of the concentration value of a nucleic acid in method herein described can be performed to provide a (T:R) value for a nucleic acid concentration in a an isolate or specimen. The T:R ratio can be defined as the ratio of the nucleic acid readout of the control sample divided by the nucleic acid readout of the antibiotic-treated sample. The T:R ratio is compared to a threshold ratio, to differentiate between the susceptible (S) and resistant (R) ratios, see CT ratios in Examples 4-5.

In particular providing a (T:R) value for a nucleic acid in an isolate or specimen can be performed by

providing a treated sample treated with the antibiotic and a control sample not treated with the antibiotic,

quantitatively detecting a control nucleic acid concentration value C in the control susceptible sample,

quantitatively detecting a treated nucleic acid concentration value T for the nucleic acid in the treated sample, and

providing a (T:R) value for the nucleic acid concentration by dividing C for the nucleic acid by T for the nucleic acid.

In methods herein described a susceptible strain of a target microorganism is identified by detecting a T:R ratio lower than a threshold.

In some embodiments the treated-reference T:R ratio, control-treated C:T ratio, or reference nucleic acid concentration value can be provided by RPKM (reads per kilobase per million mapped reads). The use of RPKM and comparison to TPM is described for example in Wagner et al 2012 [9]. In some embodiments the T:R or C:T ratio is provided by FPKM (fragments per kilobase per million), the use of FPKM is described for example in Conesa, Ana, et al. 2016 [15]. These units normalize for sequencing depth and transcript length. In some embodiments RPM (reads per million mapped reads; RPM does not normalize for transcript length) or raw sequencing read counts can be used. Typically, to calculate RPM (reads per million), the total reads from a sample are divided by 1,000,000 to obtain the “per million scaling factor”. The read counts for each gene are then divided by the “per million scaling factor” to give RPM. Also typically to calculate RPKM (for single-end RNA-seq), the RPM values are divided by the gene length in kilobases. FPKM (for paired-end RNA-seq), is calculated the same way as RPKM, taking into account that with paired-end RNA-seq, two reads can correspond to a single fragment, or, if one read in the pair did not map, one read can correspond to a single fragment as will be understood by a skilled person.

In some embodiments, to qualify for a differential concentration in the treated sample of the isolate or specimen, the difference between the C value and T value or R value and T value is statistically significant.

In preferred embodiments, the difference between the C and T value is statistically significant over the related biological variability (variability due to physiologic differences among a biological unit of a same microorganism such as between different strains of the microorganism and/or between different individual microorganism of a same strains) and/or technical variability (variability within identically performed measurements of a same biological unit due to random noise in the performance of the measuring device), more preferably over both biological and technical variability. To measure technical variability a T:R or C:T ratio is measured from a given sample multiple times with the method of choice and statistical analysis is performed on the resulting data. Technical variability would depend on the measurement method chosen. To measure biological variability, a T:R or C:T ratio is measured from multiple samples with a method that has minimal technical variability. Statistical analysis of the data comprises testing the null hypothesis that the C and T values obtained arose from the same distribution, the choice of distribution being informed according to standard statistical theory as the skilled practitioner should know [5], and rejecting the null hypothesis if the probability of generating the observed data is less than the p-value, a threshold for statistical significance chosen by the practitioner according to standards of the field. Common choices for the p-value would be 0.05, 0.025, and 0.01 (for relevant descriptions see Devore 2017 [16]). Additional description of statistical analysis used in single-molecule (digital) measurements to resolve differences between two distributions is provided in Kreutz et al 2011 [17].

In some embodiment, the reference concentration value is a total nucleic acid concentration value obtained from detection of total nucleic acid in antibiotic treated sample or in control sample (untreated with the antibiotics)

A total nucleic acid of a sample in the sense of the disclosure indicates the combined intracellular and extracellular nucleic acids in the sample. Quantification of total nucleic acids of a sample can be done following a lysis treatment of the sample and subsequent quantitative detection with methods to detect nucleic acids, after optional nucleic acid purification, including: nucleic acid amplification, gel electrophoresis, spectrophotometric detection, fluorescence detection.

Total nucleic acid of a microorganism in a sample comprise a total intracellular nucleic acid and a total extracellular nucleic acid.

The wording “total intracellular nucleic acid” as used herein refers to the portion of the total nucleic acids that are within an intact cell prior to treatment of the sample with any reagents. Accordingly, intracellular nucleic acid molecules are not able to interact will cell-impermeable reagents as will be understood by a skilled person and are detectable by detecting reagents that are impermeable to the cell. Total intracellular nucleic acid can be quantitatively detected with exemplary methods comprising filtrating the sample with a 0.2 μm filter, removing substantially all (at least 90%) extracellular nucleic acid in the sample, followed by quantifying the nucleic acids in the material retained on a filter. In addition, these methods can include incubating cell suspension with a nucleic-acid degrading reagent to degrade extracellular nucleic acids accessible to the degrading agent, followed by inactivating or removing the degrading reagent, followed by amplifying nucleic acid. For example, an RNase can be selected as a degrader to remove extracellular RNAs. The remaining RNAs are the intracellular RNA. For example, a DNase enzyme can be selected as a degrader to remove extracellular DNA. In that the remaining DNAs are the intracellular DNA.

The wording “total extracellular nucleic acid” as used herein refers to the portion of the total nucleic acids that are outside an intact cell prior to treatment of the sample with any reagents. Extracellular nucleic acid molecules are able to interact will cell-impermeable reagents as will be understood by a skilled person. Nucleic acids might be extracellular from any cells that undergo lysis, exported genetic material from a living cell, DNA in bacterial biofilms, and other means of nucleic acids exiting the cell. Methods one may use to quantify intracellular nucleic acid include cell-permeable fluorescent stains targeting nucleic acids used in combination with washing or degradation protocols to remove extracellular nucleic acids. Intracellular nucleic acids may also be quantified by subtracting extracellular nucleic acids from total nucleic acids.

In particular, in embodiments of the methods herein described wherein a total nucleic acid concentration value is detected, quantitative detection of the concentration value of a nucleic acid in method herein described can be performed to provide a value for a total nucleic acid concentration in a an isolate or specimen. The treated-total (T:tNA) ratio can be defined as the nucleic acid readout of the antibiotic-treated sample divided by the total nucleic acid readout of the sample. The T:tNA ratio is compared to a threshold, to differentiate between the susceptible (S) and resistant (R) microorganism in the sample, see Example 21.

In particular, providing a (T:tNA) value for a nucleic acid in a sample can be performed by

providing a treated sample treated with the antibiotic,

quantitatively detecting a total nucleic acid concentration value tNA in the sample treated or untreated with antibiotics,

quantitatively detecting a treated nucleic acid concentration value T for the nucleic acid in the treated sample, and

providing a (T:tNA) value for the nucleic acid concentration by dividing T for the nucleic acid by tNA for the nucleic acid.

In methods herein described a susceptible strain of a microorganism is identified by detecting a T:tNA lower than a threshold.

In some embodiments the T:tNA ratio can be provided by RPKM (reads per kilobase per million mapped reads). The use of RPKM and comparison to TPM is described for example in Wagner et al 2012 [3]. In some embodiments the T:tNA ratio is provided by FPKM (fragments per kilobase per million), the use of FPKM is described for example in Conesa, Ana, et al. 2016 [4]. These units normalize for sequencing depth and transcript length. In some embodiments RPM (reads per million mapped reads; RPM does not normalize for transcript length) or raw sequencing read counts can be used. Typically, to calculate RPM (reads per million), the total reads from a sample are divided by 1,000,000 to obtain the “per million scaling factor”. The read counts for each gene are then divided by the “per million scaling factor” to give RPM. Also, typically to calculate RPKM (for single-end RNA-seq), the RPM values are divided by the gene length in kilobases. FPKM (for paired-end RNA-seq), is calculated the same way as RPKM, taking into account that with paired-end RNA-seq, two reads can correspond to a single fragment, or, if one read in the pair did not map, one read can correspond to a single fragment as will be understood by a skilled person.

In some embodiments, to qualify for a differential concentration in the treated sample of the isolate or specimen, the difference between the T value and tNA value is statistically significant.

In preferred embodiments, the difference between the T and tNA value is statistically significant over the related biological variability (variability due to physiologic differences among a biological unit of a same microorganism such as between different strains of the microorganism and/or between different individual microorganism of a same strains) and/or technical variability (variability within identically performed measurements of a same biological unit due to random noise in the performance of the measuring device), more preferably over both biological and technical variability. To measure technical variability, a T:TNA ratio is measured from a given sample multiple times with the method of choice and statistical analysis is performed on the resulting data. Technical variability would depend on the measurement method chosen. To measure biological variability, a T:TNA ratio is measured from multiple samples with a method that has minimal technical variability. Statistical analysis of the data comprises testing the null hypothesis that the C and T values obtained arose from the same distribution, the choice of distribution being informed according to standard statistical theory as the skilled practitioner should know [5], and the practitioner rejects the null hypothesis if the probability of generating the observed data is less than the p-value, a threshold for statistical significance chosen by the practitioner according to standards of the field. Common choices for the p-value would be 0.05, 0.025, and 0.01 (for relevant descriptions see Devore 2017 [5]. Additional description of statistical analysis used in single-molecule (digital) measurements to resolve differences between two distributions is provided in Kreutz et al 2011 [6]

In some embodiments herein described, a susceptible threshold and a resistant threshold can be defined. There are various methods to define susceptible and resistant threshold as will be understood by a person skilled in the art.

In some of the embodiments for detecting accessible nucleic acids, such as in the absence of a lysis treatment or in the presence of a lysis treatment of the antibiotic treated sample following pre-lysis mechanical separation of the microorganism, a susceptible threshold and a resistant threshold can be calculated. The susceptibility or resistance of the microorganism is determined based on whether the obtained nucleic acid concentration ratio is above or below these thresholds.

As a person skilled in the art will understand, these threshold pairs are specific to each combination of bacterial species, antibiotic, concentration of antibiotic, the specific embodiment of the methods herein described, but independent of the number of samples included in the training sets.

In some embodiments, a susceptible threshold and a resistant threshold can be calculated as follows. Two sets of training sample bacteria are prepared, one with known antibiotic susceptibility and one with known antibiotic resistance, to serve as training sets, i.e. a susceptible training set and a resistant training set. Each set can have at least three training samples, preferably at least five training samples, preferably at least ten training samples, preferably at least twenty training samples, preferably at least fifty training samples, or preferably at least one hundred training samples. The susceptibility and resistance of the two training sets of sample bacteria can be pre-determined by an AST method recognized by a gold standard (e.g. the microdilution broth assay) such as by Centers for Disease Control and Prevention, by Clinical & Laboratory Standards Institute or by Food and Drug Administration or other professional organizations such as Infectious Disease Society of America as will be understood by a skilled person.

Two cutoff percentages will be designated, one referred to as “purity cutoff percentage” and the other referred to as “sensitivity cutoff percentage”. The purity cutoff percentage can be any value of at least 50% up to 100%. In particular, the purity cutoff percentage can be a value equal to or greater than 80%, 85%, 90%, 93%, 95%, 96% 97%, 98%, or 99%. The sensitivity cutoff percentage can be any value of at least 50% up to 100%. In particular, the sensitivity cutoff percentage can be a value equal to or greater than 50%, 60%, 70%, 80%, 85%, 90%, 93%, 95%, 96% 97%, 98%, or 99%.

For a given susceptible threshold, a purity percentage of that threshold is the percentage of true susceptible organisms found among organisms determined as susceptible by the method. For example, if 100 organisms were determined to be susceptible by the method herein described, and 97 of these organisms are true susceptible, then susceptible purity percentage is 97%.

For a given resistant threshold, a purity percentage is the percentage of true resistant organisms found among organisms determined as resistant by the method herein described for a given resistant threshold. For example, if 100 organisms were determined to be resistant by the method herein described, and 97 of these organisms are true resistant, then resistant purity percentage is 97%.

For a given susceptible threshold, a sensitivity percentage is the percentage of all susceptible organisms in the training set that are determined as susceptible by the method. For example, if the training set has 100 true susceptible organisms, and 96 of them were determined to be true susceptible by the method herein described, the sensitivity percentage is 96%.

For a given resistant threshold, a sensitivity percentage is the percentage of all resistant organisms in the training set that are determined as resistant by the method herein described.

The desired purity cutoff percentage and the desired sensitivity percentages can be selected based on current regulatory guidelines such as those set by the Food and Drug Administration. In addition, purity cutoff percentage and the sensitivity percentages can be selected based on commercial and business considerations as will be understood by a person skilled in the art.

A nucleic acid concentration ratio (“NACR”) is then measured using the embodiments herein described for each of the training sample in the susceptible training set and the resistant training set with a given choice of antibiotic, an antibiotic concentration and antibiotic exposure time. All the obtained NACRs will be ranked in descending order (largest to smallest).

The following steps are performed to calculate the susceptible threshold.

First is to find the lowest NACR whose purity percentage is greater than or equal to the purity cutoff percentage. For any NACR value between the lowest NACR and the largest NACR, a purity percentage is calculated by first counting the number of susceptible training samples whose NACR is greater than or equal to the given NACR, then counting the number of training samples—including both the susceptible and resistant training samples—whose NACR is greater than or equal to the given NACR, and lastly dividing the former count by the latter count.

If no such NACR is found with a purity percentage greater than or equal to the purity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method can be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

Once the lowest NACR is found, the sensitivity percentage of that NACR will be calculated. The sensitivity percentage is calculated by first counting the number of susceptible training samples whose NACR is greater than or equal to the lowest NACR, then counting the total number of susceptible training samples in the susceptible training set, and then dividing the former count by the latter count.

If the lowest NACR found does not have a sensitivity percentage above the sensitivity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

The lowest NACR whose purity percentage and sensitivity percentage are above the respective cutoff percentages will serve as the susceptibility threshold. Accordingly, any given microorganism with unknown susceptibility or resistance having a NACR value above the susceptible threshold will be identified as “susceptible”.

In some embodiments, the calculation of the susceptibility threshold described above can be repeated with higher purity cutoff percentages and/or higher susceptibility cutoff percentages.

The following steps are performed to calculate the resistant threshold.

First is to find the highest NACR whose purity percentage is greater than or equal to the purity cutoff percentage. For any NACR value between the lowest NACR and the largest NACR, a purity percentage is calculated by first counting the number of susceptible training samples whose NACR is less than or equal to the given NACR, then counting the number of training samples—including both the susceptible and resistant training samples—whose NACR is less than or equal to the given NACR, and lastly dividing the former count by the latter count.

If no such NACR is found with a purity percentage greater than or equal to the purity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

Once the highest NACR is found, the sensitivity percentage of that NACR will be calculated. The sensitivity percentage is calculated by first counting the number of susceptible training samples whose NACR is less than or equal to the highest NACR, then counting the total number of susceptible training samples in the susceptible training set, and then dividing the former count by the latter count.

If the highest NACR found does not have a sensitivity percentage above the sensitivity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

The highest NACR whose purity percentage and sensitivity percentage are above the respective cutoff percentages will serve as the resistant threshold. Accordingly, any given microorganism with unknown susceptibility or resistance having a NACR value below the resistant threshold will be identified as “resistant”.

In some embodiments, the calculation of the resistant threshold described above can be repeated with higher purity cutoff percentages and/or higher susceptibility cutoff percentages.

In some of the embodiments for detecting inaccessible target nucleic acids, such as in presence of a lysis treatment of the antibiotic treated sample following pre-lysis separation of nucleic acid from the microorganism, a susceptible threshold and a resistant threshold can be calculated. The susceptibility or resistance of the microorganism is determined based on whether the obtained nucleic acid concentration ratio is above or below these thresholds.

Similar to the steps described above with reference to the embodiments for detecting accessible target nucleic acids, two sets of training sample bacteria are prepared, one with known antibiotic susceptibility and one with known antibiotic resistance, to serve as training sets, i.e. a susceptible training set and a resistant training set. Each set can have at least three training samples, preferably at least five training samples, preferably at least ten training samples, preferably at least twenty training samples, preferably at least fifty training samples, or preferably at least one hundred training samples. The susceptibility and resistance of the two training sets of sample bacteria can be pre-determined by an AST method recognized by a gold standard (e.g. the microdilution broth assay) such as by Centers for Disease Control and Prevention, by Clinical & Laboratory Standards Institute or by Food and Drug Administration or other professional organizations such as Infectious Disease Society of America as will be understood by a skilled person.

A “purity cutoff percentage” and a “sensitivity cutoff percentage” as described above will be designated. The desired purity cutoff percentage and the desired sensitivity percentages can be selected based on current regulatory guidelines such as those set by the Food and Drug Administration. In addition, purity cutoff percentage and the sensitivity percentages can be selected based on commercial and business considerations as will be understood by a person skilled in the art.

The purity cutoff percentage can be any value of at least 80% up to 100%. In particular, the purity cutoff percentage can be a value equal to or greater than 80%, 85%, 90%, 93%, 95%, 96% 97%, 98%, or 99%. The sensitivity cutoff percentage can be any value of at least 50% up to 100%. In particular, the sensitivity cutoff percentage can be a value equal to or greater than 50%, 60%, 70%, 80%, 85%, 90%, 93%, 95%, 96% 97%, 98%, or 99%.

A nucleic acid concentration ratio (“NACR”) is then measured using the embodiments herein described for each of the training sample in the susceptible training set and the resistant training set with a given choice of antibiotic, an antibiotic concentration and antibiotic exposure time. All the obtained NACRs will be ranked in descending order (largest to smallest).

The following steps are performed to calculate the susceptible threshold.

First is to find the highest NACR whose purity percentage is greater than or equal to the purity cutoff percentage. For any NACR value between the lowest NACR and the largest NACR, a purity percentage is calculated by first counting the number of susceptible training samples whose NACR is less than or equal to the given NACR, then counting the number of training samples—including both the susceptible and resistant training samples—whose NACR is less than or equal to the given NACR, and lastly dividing the former count by the latter count.

If no such NACR is found with a purity percentage greater than or equal to the purity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method can be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

Once the highest NACR is found, the sensitivity percentage of that NACR will be calculated. The sensitivity percentage is calculated by first counting the number of susceptible training samples whose NACR is less than or equal to the highest NACR, then counting the total number of susceptible training samples in the susceptible training set, and then dividing the former count by the latter count.

If the highest NACR found does not have a sensitivity percentage above the sensitivity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

The highest NACR whose purity percentage and sensitivity percentage are above the respective cutoff percentages will serve as the susceptibility threshold. Accordingly, any given microorganism with unknown susceptibility or resistance having a NACR value below the susceptible threshold will be identified as “susceptible”.

In some embodiments, the calculation of the susceptibility threshold described above can be repeated with higher purity cutoff percentages and/or higher susceptibility cutoff percentages.

The following steps are performed to calculate the resistant threshold.

First is to find the lowest NACR whose purity percentage is greater than or equal to the purity cutoff percentage. For any NACR value between the lowest NACR and the largest NACR, a purity percentage is calculated by first counting the number of susceptible training samples whose NACR is greater than or equal to the given NACR, then counting the number of training samples—including both the susceptible and resistant training samples—whose NACR is greater than or equal to the given NACR, and lastly dividing the former count by the latter count.

If no such NACR is found with a purity percentage greater than or equal to the purity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

Once the lowest NACR is found, the sensitivity percentage of that NACR will be calculated. The sensitivity percentage is calculated by first counting the number of susceptible training samples whose NACR is greater than or equal to the highest NACR, then counting the total number of susceptible training samples in the susceptible training set, and then dividing the former count by the latter count.

If the lowest NACR found does not have a sensitivity percentage above the sensitivity cutoff percentage, then a threshold that satisfies the desired cutoff percentage is unavailable, and the method will be modified by modifying the antibiotic exposure time, changing the antibiotic concentration, selecting a different cutoff percentage, and/or modifying the enhancers.

The lowest NACR whose purity percentage and sensitivity percentage are above the respective cutoff percentages will serve as the resistant threshold. Accordingly, any given microorganism with unknown susceptibility or resistance having a NACR value above the resistant threshold will be identified as “resistant”.

In some embodiments, the calculation of the resistant threshold described above can be repeated with higher purity cutoff percentages and/or higher susceptibility cutoff percentages.

In lieu of the algorithm described above, there exist other mathematical algorithms to find a NACV threshold, some equivalent to each other when applied to 1-dimensional data. The algorithms all take as their input the same training set of NACVs and their corresponding gold-standard AST calls as the above algorithm. These other mathematical algorithms include algorithms in which a receiver operating characteristic curve is calculated, and thresholds are chosen that maximize the area under the curve (AUC), maximize the sensitivity, maximize the specificity, or are found by a weighted sum or other combination of these metrics; supervised machine learning techniques for binary or multi-class classification, including support vector machines with soft margins, linear regression, logistic regression, and ordinal regression; probabilistic methods in which the distributions of susceptible and resistant samples are each modeled as probability density functions of random variables, and then thresholds are drawn which correspond to a probability, chosen by the practitioner as desired, that incorrect calls will be made; clustering algorithms. Descriptions of these algorithms are found in reference [18].

The terms “statistical test”, “machine learning technique”, and “machine learning algorithm” used herein refer to any one of a variety of models and algorithms, or combination of such models and algorithms, described in the literature and known to the skilled person which can be employed at any step in the disclosed methods herein requiring one to classify observations from numerical or categorical data; that is, to predict whether observations arose from a certain class of entity. To perform classifications from data, one can employ statistical tests, algorithms that assume an underlying statistical models and give the probability or likelihood of summary statistics. In addition, or as an alternative, one can employ machine learning algorithms, which are algorithms that map data to the classification output, sometimes assuming an underlying statistical model. Example steps \ that use statistical tests or machine learning techniques include the calling of well loading status during sample partitioning, the calling of antibiotic susceptibility by each accessibility AST embodiment, and the creation of thresholds for antibiotic susceptibility calls calculated from prior experiments. Each statistical test or machine learning technique's performance varies depending on the way a particular embodiment of accessibility AST generates its data, and some tests are not appropriate for some situations. Some tests are special cases of a more generalized, more complicated test. Using a more complicated algorithm to analyze a simple data set will be equivalent is not necessary. These unsupervised and supervised machine learning algorithms and statistical tests include: any univariate or multivariate, parametric or non-parametric, one-sided or two-sided, paired or independent, frequentist or Bayesian statistical model and test (t-tests, multiple t-tests, analysis of variance (ANOVA), repeated measures ANOVA, one-way ANOVA, multivariate analysis of variance (MANOVA), analysis of covariance, Pearson's r test, Spearman's r, McNemar test, Friedman test, Durbin test, Fisher's exact test, Boschloo's test, Barnard's test, Chi-square test, the sign test, the exact Z-pooled and Z-unpooled tests, Kruskal-Wallis test, Mann-Whitney U/Wilcoxon rank-sum test, Wilcoxon signed-rank test, Kolmogorov-Smirnov test, bootstrapping, Gaussian and other parametric mixture models, multilevel models, Bayesian hierarchical models); regression analysis (linear regression, multiple regression, gradient descent, ordinary least squares regression, logistic regression, probit regression, generalized linear regression, non-linear regression, mixed effects models, measurement error models, Bayesian regression, ridge regression, LASSO, locally-weighted linear regression, multivariate adaptive regression splines, nonparametric regression); times series analysis (autoregressive models, autoregressive moving average models, autoregressive integrated moving average models, stochastic processes, branching processes, Gaussian processes, survival analysis, Kaplan-Meier estimate, proportional hazards models, Cox proportional hazard model, log-rank test); cluster analysis (k-means clustering, k-medoids clustering, partitioning around medoids clustering, nearest neighbors clustering, hierarchical clustering, agglomerative hierarchical clustering, divisive hierarchical clustering, density-based spatial clustering of applications with noise, stochastic network embeddings); matrix factorization techniques (principle components analysis, non-negative matrix factorization, singular value decomposition, collaborative filtering, spectral clustering), artificial neural networks (including “deep learning”, deep artificial neural networks); other supervised classification algorithms (decision trees, classification and regression trees, random forests, support vector machines), generative or Bayesian probability models (naïve Bayes classifiers, Bayesian/belief/probabilistic graphical networks/models); general adversarial networks, and reinforcement learning.

Nucleic acid concentration ratio can be obtained by dividing nucleic acid concentration values. It can also be described as a mathematically equivalent operation, for example, for an exponential amplification process by taking a difference of concentration parameters, such as the difference of Cq values (cycle threshold values) from qPCR. As described herein, a nucleic acid concentration value can be calculated from a Cq by equation (1). A ratio can be computed from two nucleic acid concentration values from Cq measurements as seen in equation (2). This expression can be rearranged to include a difference term, as seen in equation (3).

Nucleic acid concentration value=2{circumflex over ( )}(−Cq)  (1)

T:R ratio=(2{circumflex over ( )}−Cq treated)/(2{circumflex over ( )}-Cq reference)  (2)

T:R ratio=2{circumflex over ( )}(Cq reference−Cq treated)  (3)

When using concentration parameters, the concentration parameters obtained by the nucleic acid detecting method may be converted to an equivalent nucleic acid concentration value using the formulas described herein or by constructing and consulting a standard curve. Then, the nucleic acid concentration ratio can be calculated from the equivalent nucleic acid concentration values. However, as illustrated herein, NACR can be calculated directly from the concentration parameters, for example for an exponential amplification process by taking a difference of concentration parameters, such as the difference of Cq values (cycle threshold values) from qPCR, or by analogous mathematical transformations that convert reaction times (such as in term of time-to-positives) of exponential isothermal amplification directly into a ratio. For detection methods with linear dependence of concentration parameter on concentration (results of digital amplification and number of reads of the target nucleic acids obtained from high throughput sequencing), ratio of concentration parameters can be obtained directly without converting them into nucleic acids concentration values.

In preferred embodiments, the nucleic acid concentration ratio is detected as a nucleic acid concentration relative differences as will be understood by a skilled person. There are several mathematical definitions of a relative difference known to the skilled person. Nucleic acid concentration relative differences can be obtained by mathematically manipulating nucleic acid concentration values according to these definitions. The definitions of relative difference include the following:

Relative difference=(concentration reference−concentration target)÷((concentration reference+concentration target)÷2);  1)

Relative difference=(concentration reference−concentration target)÷((abs(concentration reference)+abs(concentration target))÷2;  2)

Relative difference=(concentration reference−concentration target)÷max(concentration reference,concentration target);  3)

Relative difference=(concentration reference−concentration target)÷max(abs(concentration reference),abs(concentration target));  4)

(concentration reference−concentration target)÷min(concentration reference,concentration target);  5)

(concentration reference−concentration target)÷min(abs(concentration reference), abs(concentration target)). In addition, any of these formula may be multiplied or divided by a constant real number, such as in:  6)

Relative difference=(concentration reference−concentration target)÷(concentration reference+concentration target.  7)

In all the above equations, if a Cq is used to quantify nucleic acids, then the wording “concentration reference” can be replaced by “K{circumflex over ( )}-Cq” wherever it appears, where K is a real number such as 2, 1.9, or whatever is the calculated “qPCR per cycle efficiency” according to methods identifiable by a skilled person.

In embodiments wherein the nucleic acid concentration value is expressed as a time-to-positive (TTP, a reaction time for an isothermal amplification), a nucleic acid concentration relative differences can be expressed a TTP difference value (“TTPD”), which can then be used to determine susceptibility. In particular, in those embodiments, higher concentration of nucleic acid to antibiotics-treated sample, will result in an earlier TTP relative to a reference control sample or relative to a reference sample treated under same or different exposure conditions (typically lower antibiotic concentration and/or antibiotic contacting for a shorter contacting time). (see Example 25)

In particular, in some embodiments wherein the nucleic acid concentration value is expressed as time-to-positive, the reference sample can be fully lysed and a total nucleic concentration can be determined by a total nucleic acid time-to-positive, which is then used to provide a TTPD and/or to derive a nucleic acid concentration value to be used in determining an accessible or inaccessible nucleic acid percentage as will be understood by a skilled person.

In some embodiments wherein the nucleic acid concentration value is expressed as a time-to-positive, the reference sample can be subjected to nucleic acid amplification and the related TTP can be compared with the TTP of the treated sample to provide a nucleic acid concentration relative differences in terms of TTPD.

In some embodiments of methods of the disclosure, antibiotic susceptibility in a target microorganism of the instant disclosure can comprise selecting the sample with a detected high fold-change in detected nucleic acid concentration upon antibiotic exposure. In those embodiments, the fold-change is calculated based on nucleic acid quantification results. in particular, fold-change may be computed from qPCR Cq values or from digital PCR concentrations. When representing a decreasing fold-change, it may also be visualized as the reciprocal 1/FC, see Example 3. Fold change is compared to a threshold fold change to differentiate between the susceptible (S) and resistant (R) fold changes. If the fold-change comprises a treated nucleic acid concentration value divided by a reference nucleic acid concentration value, then the fold-change is equivalent to a NACR.

A high fold change is defined as at least two folder change or higher. In particular, in some embodiments, a significant shift of fold change (larger than 4) in concentration levels can be observed within 5 min, 10 min, or 15 min of antibiotic exposure.

In preferred embodiments, the R and T value is adjusted to reduce the impact of biological variability and/or technical variability, more preferably of both biological and technical variability. Accordingly, in some embodiments, the methods herein described, further comprises normalizing the (T:R) value prior to detecting a differential concentration of nucleic acid in the treated samples.

The wording “normalizing” and “normalization” as used herein refer to adjustments of a value related to a quantified amount to account for variations. In particular normalization of a value can be performed to account for a variation in a parameter associated with the detection of the quantified amount, such as variations in an amount of starting material, variations in an amount of sample, variations in bacterial concentration of sample, variations due to biological variability and variations due to technical variability.

Normalizing the T:R value can be performed with a reference measurement of cell number, the number of samples, the volume of sample used, the concentration of sample used, the effective amount of sample used and/or a related ratio in a reference and in a treated sample. Effective amount of sample can be calculated by for example measuring the volumes and concentration of the sample used. Normalizing the T:R value can be performed by dividing the control nucleic acid concentration by a reference measurement in the control sample and dividing the treated nucleic acid concentration by the reference measurement in the treated sample. Normalizing the (T:R) value can be performed by dividing the (T:R) value by a normalization ratio. The normalization ratio for the sample can be calculated by dividing the control reference measurement by the treated reference measurement.

In some embodiments of these embodiments, the normalizing measurement is a measurement that reflects the number of target cells. For example, prior to the calculation of a CT ratio, the detected nucleic acids in the untreated control sample and the detected nucleic acids in the treated sample would be divided by a cell normalization ratio between number of target cells in the treated sample and number of target cells in the control sample which can be calculated from other measurements such as optical density, turbidity, increase in intensity of a colorimetric, fluorogenic, or luminescent metabolic indicator or a live/dead indicator, colony counting after plating, amount of pathogen-specific DNA and amount of pathogen-specific RNA as will be understood by a skilled person.

In some embodiments the number of cells of the target microorganism present in a treated or control experiment can be estimated from a number of detected pathogen-specific DNA or RNA copies. In some embodiments of methods directed to detect inaccessible nucleic acid, two detected nucleic acids from a single sample are compared. These comparisons, ideally, are between one changing nucleic acid (such as changes in accessibility with addition of a targeting nuclease) and one unchanging nucleic acid (such as DNA in a sample which is known not to undergo substantial DNA replication under incubation conditions, such as N. gonorrhoeae in MHB media). In some methods, the extracted sample can be split only for the detection step two different primer sets are used. In other methods/examples, an incubation method includes RNase, nucleic acid quantification results can be used to calculate a DNA target and an RNA target from the same sample. The ratio of inaccessibility T:R ratio (e.g. the treated RNA sample with a degrading RNase divided by the reference RNA sample with a degrading RNase) can be normalized (by division of the ratios) to a ratio of DNA T:R ratio (without a degrader targeting the DNA, this number will be equal to 1.0 in a situation without any biological or technical noise). DNA and RNA concentrations can be quantified with qPCR, digital PCR, digital isothermal amplification, real-time isothermal amplification, digital LAMP, real-time LAMP. To differentiate between DNA and RNA concentrations reactions with and without reverse transcriptase are performed. The DNA/RNA ratio can be defined as the ratio of DNA readout, such as concentration of copies/μL, divided by the RNA readout, such as concentration of copies/μL, (see Example 6).

In some embodiments the number of cells of the target microorganism present in a treated or control experiment can be estimated from a number of detected pathogen-specific DNA or RNA copies. In some methods, a reference measurement will be done of the initial detectable inaccessible target nucleic acids. This is similar to the estimation of total amount of cells, but uses a degrading treatment of nucleic acids to remove any background, nucleic acid molecules from a target microorganism. This reference sample is sometimes referred to as a time-zero reference or “t0” reference as the total amount of inaccessible target molecules before treatment begins. In some methods, the T:R ratio can be used alone, refer to Example 10 for experimental methods and data. In other methods, the t0 ratio of the untreated and treated samples can be used to normalize the comparison between the untreated and treated samples to normalize for aforementioned biological and technical variability.

In some embodiments, the number of cells of the target microorganism present in a treated or control experiment can be estimated by both the presence and number of detected pathogen-specific DNA or RNA copies, rather than only from the number of detected pathogen-specific DNA or RNA copies. In those embodiments, the presence of any amount of pathogen-specific DNA or RNA copies indicates that there is at least one individual microorganism in the sample, while the absence of pathogen-specific DNA or RNA indicates that no microorganisms were present in the sample. The concentration of one DNA or RNA molecule per sample volume can be used to define a threshold concentration for inferring the presence or absence of a cell in the sample. This threshold is increased, depending on the accuracy in nucleic acid quantification to distinguish samples containing at least one cell from those containing no cells but some false positive detections of nucleic acids.

In some embodiments of methods directed to detect accessible nucleic acid, two detected nucleic acids from a single sample are compared. Before detection, a sample may be split into two or more sub-samples. One sample may undergo a lysis treatment, to obtain a sample containing total nucleic acids from the target microorganism. The other part of the sample may not undergo any lysis treatment. Both samples can be quantified using a non-lysing amplification method such as qLAMP; only the accessible target nucleic acid molecules are detected in sample without lysis, which are compared to the total nucleic acid molecules from the sample which undergoes lysis prior to detection (see Example 21 for experimental methods and data).

In some embodiments of methods and systems herein described to detect accessible or inaccessible nucleic acid, before detection, a sample can be split into two or more sub-samples, and some sub-samples can be then processed to detect accessible or inaccessible nucleic acid following antibiotic treatment and some sub-samples can be processed to detect total nucleic acid (e.g., Examples 10, 21, Example 25 to 30, Examples 32-37 and Examples 38 to 42).

In some embodiments of methods and systems herein described to detect accessible or inaccessible nucleic acid, a sample can be split in a plurality of partitions to allow detection of accessible nucleic acid, inaccessible nucleic acid, or total nucleic acid on sub-samples as will be understood by a skilled person (see Examples 10, 21, and 38 to 42).

In those embodiments, treated samples and/or reference samples are provided by sub-samples, a reference aliquot and a treated aliquot of a sample of an isolate or specimen can be provided by splitting the sample into two or more aliquots, typically of equal amounts, wherein one or more aliquots are treated with antibiotics (treated aliquots) and other aliquots (reference aliquot) are independently untreated or treated with antibiotics for a certain amount of time. In some of those embodiments, one or more reference aliquots can be used to detect a total nucleic acid concentration. In those embodiments, and the reference aliquot can be a fully lysed aliquot created by extracting nucleic acids from an aliquot of a control sample, wherein the reference aliquot is either treated or untreated with the antibiotic (see Examples 10, 21, 38-42).

In some embodiments, wherein treated and/or reference samples are provided by sub-samples, a reference aliquot can be a reference sub-sample treated with antibiotics, In those embodiments, the methods and systems herein described can be performed on an antibiotic-treated sample which is split in one or more treated aliquots and one or more reference aliquots following antibiotic exposure at same or different contacting time (see Examples 10, 21, 38-42).

In some embodiments, wherein treated and/or reference samples are provided by sub-samples, a treated aliquot can comprise a plurality of treated aliquots treated at different exposure conditions (e.g. different contacting time and/or contacting performed at different antibiotic concentration). In those embodiments, the plurality of reference aliquots can further be provided comprising for example a plurality of control aliquot wherein detecting is performed from each control aliquot following exposure conditions corresponding to the exposure conditions of the contacting of a corresponding treated aliquot performed however in absence of the antibiotic. The plurality of reference aliquots can in addition or in the alternative comprise a plurality of reference sub-samples treated with antibiotics which can be used to provide total nucleic acid concentrations (in particular with same exposure conditions compared to a corresponding treated aliquot) or a reference nucleic acid concentration value (in particular with same exposure conditions compared to a corresponding treated aliquot but for different contacting time and/or different concentration of antibiotic) (see Example 10, 21, 25-30 and Examples 38-42).

In embodiments, wherein the treated sample and reference samples are provided by treated aliquots and reference aliquots which are antibiotic treated, the method herein described can be performed on an antibiotic-treated sample which is split in aliquots following antibiotic exposure at the same or different contacting time as will be understood by a skilled person upon reading of the present disclosure. In those embodiments, using only a single sample or aliquot of the original sample during the exposure to antibiotic which is further split following the contacting reduces the challenges of fluid handling and metering as will be understood by a skilled person. (see Example 10, 21, 25-30, and Examples 38-42)

In some embodiments, quantitatively detecting T:R, C:T, or total nucleic acid can be performed on a treated sample and corresponding control sample under several sets of conditions (e.g. varying treatment times, different experimental settings and/or using a plurality of isolates or specimen and/or a plurality of related control and/or treated sample) to provide a nucleic acid concentration pattern for the nucleic acid in each treated and corresponding control samples under each set of conditions. In those embodiments, the differential concentration of the nucleic acid is detected with respect to the corresponding gene expression pattern or nucleic acid concentration value according to approaches identifiable by a skilled person upon reading of the present disclosure.

In some embodiments of the method of the instant disclosure, the target microorganism is a slow growing microorganism, a microorganism with a transcriptome which is not characterized and/or a microorganism that lacks a transcriptional SOS response to DNA damage.

The term “slow growing” as used herein indicates an organism with a doubling time longer than 30 minutes.

In some embodiments of the methods of the instant disclosure, the antibiotic is an antibiotic which interacts directly or indirectly with the cell envelope, cell envelope biosynthesis, cell structural remodeling, or overall cell integrity. For example, beta lactam antibiotics and antibiotic agents that contain a beta-lactam ring in their molecular structures. Including penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. Exemplary beta-lactam antibiotics include penicillin, ceftriaxone, cefixime, ampicillin, amoxicillin, meropenem, imipenem, and ertapenem.

In some of these embodiments, the antibiotic for treating the sample herein described, the concentration of the antibiotic can be provided at a concentration between 0.015 microgram/mL and 16.0 microgram/mL.

In some of these embodiments, the beta-lactam is penicillin. In some of these embodiments, the concentration of antibiotic used during exposure or treatment can be any concentration between the susceptible and resistant MIC breakpoints of the target organism. For example, for exposure or treatment of Ng with ciprofloxacin, the concentration of antibiotic used could be any concentration≥0.06 microgram/mL (the susceptible MIC breakpoint for penicillin for Neisseria gonorrhoeae) and ≤2.00 microgram/mL (the resistant MIC breakpoint for penicillin for Neisseria gonorrhoeae). In some embodiments, for example when faster responses are desired, higher than breakpoint concentrations can be used.

In some embodiments, methods herein described can be performed on a treated sample only, without need of a control sample and related incubation. An incubation method can include a degrading molecule, such as an RNase, to degrade accessible RNAs (or complementarily, DNase to degrade accessible DNAs) followed by nucleic acid quantification of the accessible nucleic acids. Quantification can be performed on nucleic acids split after a single, antibiotic-treated sample. The reference measurement of an unchanging nucleic acid may refer to DNA, unchanging mRNA transcript, or the initial total amount of that nucleic acid target prior, during, or after treatment can be used to calculate a T:R ratio. The treated (T) part of that ratio is a nucleic acid target and the reference (R) refers to the reference target nucleic acid, which may differ in primer target, nucleic acid type (e.g. DNA or RNA), which can be measured from the same sample for nucleic acid detection. For example, a DNA/RNA ratio alone can be used to differentiate susceptible and resistant strains. See Example 6.

In some embodiments of methods herein described a specimen does not need to be split equally between treated and control sample. For example, when DNA/RNA ratio is used to predict susceptibility. This aforementioned DNA/RNA ratio can be used as a normalization factor or as a ratio for the susceptibility readout. Advantages of using two nucleic acid targets of amplification in the same sample include unequal numbers of cells between the reference sample and the treated sample that may occur. When the quantifiable nucleic acids are targeted from the same extraction of the treated sample, there is no longer a concern that the sample containing microorganisms. See Example 6, only the treated sample is required for the susceptibility call.

In some embodiments, nucleic acid extraction methods are used to inactivate nucleases. Extraction methods include but are not limited to Epicentre DNA Extraction Buffer and Zymo Research Viral DNA/RNA kit. In the instance in which nuclease treatments are used, such as the aforementioned exogenous degrading nucleases or sample pre-treatment of host nucleic acids, these degraders are desired to be inactivated prior to nucleic acid detection, as they may interfere with the measurement. The extraction methods described in experiments containing nucleases (DNaseI, RNaseA, or RNase cocktail) are inactivated with the corresponding nucleic acid extraction and lysis treatment of the sample, including the Lucigen DNA extraction buffer (DEB) and the Zymo Viral DNA/RNA lysis kit through the use of proteases and heat (DEB) and the use of denaturing agents in the Zymo kits, as is the case with the examples provided. If other nucleic acid extraction methods are used, then the method must be assessed for the ability to also inactivate the exogenous nucleases or add an additional treatment or step, such as including inhibitors, proteases, or quenching molecules to stop the nucleases. Other options include conjugation of nucleases to beads to enable physical, magnetic, or other targeted separation of the nucleases from the sample prior to nucleic acid quantification.

In some embodiments of the methods herein described, the target microorganism is N. gonorrhoeae.

Neisseria gonorrhoeae is one type of proteobacteria that causes the sexually transmitted genitourinary infection gonorrhea, as well as other forms of gonococcal disease including oropharyngeal gonorrhea, rectal gonorrhea, disseminated gonococcemia, gonococcal septic arthritis, and gonococcal ophthalmia neonatorum. The term “Neisseria gonorrhea” includes all strains of N. gonorrhoeae identifiable by a person skilled in the art. Neisseria gonorrhoeae also includes genetic variants of different strains. One may determine whether the target organism is N. gonorrhoeae by a number of accepted methods, including sequencing of the 16S ribosomal RNA (rRNA) gene, as described in Chakravorty et al (2007) for N. gonorrhoeae. [19].

In some embodiments of the method herein described the target microorganism is of the family Enterobacteriaceae, for example Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp. Organisms in the family Enterobacteriaceae are Gram-negative bacteria that can cause multiple types of infections (such as urinary tract infections, enteritis, dysentery, pneumonia, meningitis, bacteremia, and sepsis), especially in healthcare settings.

In some embodiments of the methods of the instant disclosure wherein the target microorganism is N. gonorrhoeae, the time period of contacting the sample with an antibiotic is shorter than the doubling time of the N. gonorrhoeae strain in the sample. For example, for conditions with N. gonorrhoeae doubling time of 45 minutes, 1 hour, 1.5 hours, or 2 hours, antibiotic exposure contacting time could be less than the time indicated in Table 1 below.

TABLE 1 time of contacting N. gonorrhoeae with antibiotic 45 minute 60 min 90 min 120 doubling doubling doubling doubling factor X contacting time, less than (minutes): 1 45 60 90 120 0.75 33.75 45 67.5 90 0.5 22.5 30 45 60 0.35 15.75 21 31.5 42 0.25 11.25 15 22.5 30 0.2 9 12 18 24 0.15 6.75 9 13.5 18 0.1 4.5 6 9 12 0.075 3.375 4.5 6.75 9 0.05 2.25 3 4.5 6

In methods of the instant disclosure wherein the target microorganism is N. gonorrhoeae, incubation of a sample with an antibiotic can be performed at a temperature such that a physiological response to the antibiotic is generated in N. gonorrhoeae. The contacting is performed typically in an incubation temperature at 37° C., in an incubation temperature within the range of 36-38 degrees ° C., in an incubation temperature within the range of 35-39 degrees ° C.

In methods of the instant disclosure wherein the target microorganism is incubated with antibiotics, the incubation can be performed by adding antibiotics to the microorganism and incubating the sample under certain condition preferably following and/or upon contacting the sample with a treatment media designed to support physiological processes of the target microorganism, and/or enable or accelerate DNA replication and translation, and/or enable or accelerate cell wall biosynthesis, maintenance, or repair, and/or maintain cellular uniformity and homogeneity in suspension, and/or promote interaction of the target microorganism and antibiotic herein described.

In some embodiments of the methods of the instant disclosure, quantitatively detecting a nucleic acid concentration value can be performed using probes specifically targeting any one sequence of the nucleic acids of the target microorganism.

The term “probe” as described herein indicates a molecule or computer support tool capable of specifically detecting a target molecule such as nucleic acids herein described. The wording “specific” “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, together with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and the second molecule with other molecules that may be present. Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions and additional interactions identifiable by a skilled person. The wording “specific” “specifically” or “specificity” as used herein with reference to a computer supported tool, such as a software indicates a tool capable of identifying a target sequence (such as the nucleic acids of the target organism herein described) among a group of sequences e.g. within a database following alignment of the target sequence with the sequences of the database. Exemplary software configured to specifically detect target sequences comprise Primer-3, PerlPrimer and PrimerB last.

In some embodiments of the methods of the instant disclosure, the probe specific for the sequence of the target nucleic acid is selected from a primer having a sequence specific for the target nucleic acid, or an antibody specific for the target nucleic acid.

In particular, probes usable in methods herein described can include primers for nucleic acid amplification reactions (such as polymerase chain reaction (PCR), loop mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase reaction (RPA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), and additional reactions identifiable by a skilled person), including digital single molecule versions of these reactions and including real-time versions of these reactions, molecular beacons that include dyes, quenchers, or combinations of dyes and quenchers.

Nucleic acid probes preferably have sequences that complementarily bind to the DNA and/or RNA sequences of the nucleic acids from target organisms described herein, and can be used to target RNA molecules directly, or DNA molecules that result, for example, from reverse transcription of the target RNA molecules (such molecules may be referred to as cDNA). In embodiments of the present disclosure when two polynucleotide strands, sequences or segments are noted to be binding to each other through complementarily binding or complementarily bind to each other, this indicate that a sufficient number of bases pairs forms between the two strands, sequences or segments to form a thermodynamically stable double-stranded duplex, although the duplex can contain mismatches, bulges and/or wobble base pairs as will be understood by a skilled person.

In embodiments herein described primers and/or other nucleic acid probes can be designed to complementarily bind nucleic acids of the target microorganism herein described with methods described in [20].

Probes usable in methods herein described include probes used in guiding CRISPR-based detection of Nucleic acids. e.g. CRISPR-associated protein-9 nuclease; CRISPR-associated nucleases. An example of a CRISPR-based method is described in references [21] [22] [23]. Such probes can be synthesized using naturally occurring nucleotides including deoxylnosine, or include unnatural nucleotides such as locked nucleic acid (LNA). Probes can comprise dyes, quenchers, or combinations of dyes and quenchers attached to the probe. Hybridization probes, including those used in fluorescent in situ hybridization and hybridization chain reaction. Probes can also comprise electrochemically active redox molecules attached to the probe. Probes can be provided in a dry state. Probes can also include probes bound to beads, such beads may be fluorescently labeled. Probes can also include probes bound to nanoparticles, such nanoparticles may include gold nanoparticles. Probes can include probes disposed in arrays of wells with volumes less than 50 microliters, and/or wells within plastic substrates.

In some embodiments, of the methods of the instant disclosure, quantitatively detecting of a nucleic acid can be performed by detecting a detectable portion thereof. Exemplary detectable portions comprise to regions of at least 14 base pair, at least 16 base pair, at least 18 base pair, at least 19 base pair, at least 20 base pair, at least 21 base pair, at least 22 base pair, at least 23 base pair, at least 24 base pair, at least 30 base pair, at least 40 base pair, at least 50 base pair, at least 60 base pair, at least 70 base pair, at least 80 base pair, at least 90 base pair, or at least 100 base pair, The specific portion can be identified by a skilled person based on the length of the nucleic acid to be detected as will be understood by a skilled person.

In some embodiments of the methods of the instant disclosure, the methods comprise detecting whether there is an increase in the nucleic acid concentration value, in a sample treated with an antibiotic with respect to a sample not treated with antibiotic.

In particular in embodiments of the methods of the instant disclosure, the methods comprise detecting whether there is an upshift of a detected increase in a treated nucleic acid concentration value detected following treatment with antibiotic with respect to an untreated nucleic acid concentration value in absence of antibiotic treatment.

In some embodiments of the methods of the instant disclosure using any target organisms herein described, the sample can be stored until sample preparation and analysis, for example at room temperature, 4° C., −20° C., or −80° C., as appropriate, identifiable by those skilled in the art. When biological specimens are stored, they ideally remain equivalent to freshly-collected specimens for the purposes of analysis. In some embodiments, of the methods of the instant disclosure using any one of target organisms herein described, the sample can be pre-incubated with growth media for a short period of time to increase the number of viable bacterial cells or to increase the level of nucleic acids in such cells. The temperature and media for such pre-incubation can be performed as described herein for incubation. The duration of such pre-incubation can range, for example, from 5 minutes to 20 minutes to 1 hour to 2 hours.

In some embodiments methods and systems herein described, the detecting can be performed in presence of a lysis treatment of the antibiotic treated sample targeting the microorganism; and in presence of sample separation performed by mechanical separation of the nucleic acid (extracellular fraction) from the microorganism of the sample, or by mechanical separation of the microorganisms (cellular fraction) from the nucleic acid of the sample.

In some embodiments methods and systems herein described, the quantitatively detecting can be performed in presence of a lysis treatment of the antibiotic treated sample targeting the microorganism; and in presence of pre-lysis sample separation performed by mechanical separation of the nucleic acid (extracellular fraction) from the microorganism of the sample, or by mechanical separation of the microorganisms (cellular fraction) from the nucleic acid of the sample.

In some of those embodiments, the method can comprise

-   -   contacting the sample with an antibiotic to provide an         antibiotic treated sample,     -   performing mechanical separation of the microorganism from the         nucleic acid in the antibiotic treated sample to provide an         antibiotic treated separated sample comprising extracellular         nucleic acid of the microorganism; and     -   quantitatively detecting in the antibiotic treated separated         sample the extracellular nucleic acid of the microorganism to         obtain an detected antibiotic treated nucleic acid concentration         value of the microorganism in the antibiotic treated sample,         the quantitatively detecting performed in presence of a lysis         treatment of the antibiotic treated sample targeting the         microorganism,

In other embodiments, the method can comprise

-   -   contacting the sample with an antibiotic to provide an         antibiotic treated sample,     -   performing mechanical separation of nucleic acid from         microorganism in the antibiotic treated sample to provide an         antibiotic treated separated sample comprising the intracellular         nucleic acid of the microorganism; and         quantitatively detecting in the antibiotic treated separated         sample the intracellular nucleic acid of the microorganism to         obtain an detected antibiotic treated nucleic acid concentration         value of the microorganism in the antibiotic treated sample.

In some embodiments the mechanical separation is performed by centrifugation resulting in the pelleting/sedimentation of the microorganism component while the nucleic acid component will be suspended in the liquid.

In some preferred embodiments, the mechanical separation is performed by filtration with the nucleic acid comprised in the filtrated and the microorganism comprised in the retentate. In more preferred embodiments, the mechanical separation can be performed by filtration with centrifugation and/or under vacuum to increase the flow rate of the sample through the filter In some of those embodiments filtration can be performed with filters of a solid, gelatinous, granular, woven, or other porous material containing pores no larger than a desired size; the just-mentioned filtration with or without a subsequent wash of a liquid substance; filtration using porous substances that specifically and reversibly bind bacteria using covalent or non-covalent chemical bonds, or other filtration techniques existing in scientific literature as will be known to the skilled person.

In some of those embodiments, the method further comprises concurrently or after the contacting and before the detecting performing an enhancing treatment of the antibiotic treated sample. In particular, any enhancers described herein can be used, preferably surfactants, and more preferably non-ionic and/or zwitterionic detergents.

In some embodiments, the methods and systems comprising performing mechanical separation of the microorganism from the nucleic acid in the antibiotic treated sample the quantitatively detecting can be performed using any lytic or non-lytic amplification reactions such as polymerase chain reaction (PCR) including quantitative real-time PCR (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), and multiplexed versions of such PCR techniques; and isothermal techniques such as loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase reaction (RPA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA) for whole genome amplification (WGA), nicking enzyme amplification reaction (NEAR), and additional reactions identifiable by a skilled person, and preferably digital versions of these isothermal amplifications; hybridization such as nucleic acid microarrays and fluorescence in situ hybridization; optical measurements such as absorbance measurements such as UV/vis spectroscopy and fluoroscopy (e.g. quantification of nucleic acids by a nucleic-acid specific fluorescent dye); electrochemical measurements of nucleic acid concentration, such as those that measure redox reaction currents enabled by binding of nucleic acids to a functionalized electrode and additional nucleic acid quantification techniques identifiable to a skilled person.

In some embodiments, the methods and systems comprising performing mechanical separation of the microorganism from the nucleic acid in the antibiotic treated sample, the method can further comprise detecting a nucleic acid concentration ratio and preferably a differential concentration value, in the sample by comparing the detected antibiotic treated nucleic acid concentration value with a detected reference nucleic acid concentration value of a nucleic acid concentration parameter of the microorganism in the sample.

In particular, in embodiments of the methods and systems comprising performing mechanical separation of the microorganism from the nucleic acid in the antibiotic treated sample, the detected reference nucleic acid concentration value is a control nucleic acid concentration value and/or a total nucleic acid concentration value.

In those embodiments, the control nucleic acid concentration value is obtained by detecting a nucleic acid concentration for the nucleic acid of the microorganism in a control sample of the isolate or specimen comprising the microorganism preferably from the same isolate or specimen of the initial sample used to provide the antibiotic treated sample. In particular, detecting a nucleic acid concentration for the nucleic acid of the microorganism in a control sample to provide the control nucleic acid concentration value for the nucleic acid of the microorganism is performed

-   -   in presence of a lysis treatment of the control sample following         pre-lysis mechanical separation of nucleic acid from the         microorganism in the control sample to provide a separated         control sample comprising intracellular nucleic acid of the         microorganism, when the separated antibiotic treated sample         comprises intracellular nucleic acid of the microorganism; or     -   in presence of a lysis treatment of the control sample following         pre-lysis separation of microorganism from the nucleic acid in         the antibiotic treated sample, to provide a separated control         sample comprising extracellular nucleic acid of the         microorganism when the separated antibiotic treated sample         comprising extracellular nucleic acid of the microorganism,

In embodiments wherein the detected reference nucleic acid concentration value comprises a total nucleic acid concentration value, the total nucleic acid concentration value can be obtained by detecting a nucleic acid concentration for the nucleic acid of the microorganism in a treated or untreated reference sample of the isolate or specimen comprising the microorganism preferably from the same isolate or specimen of the initial sample used to provide the antibiotic treated sample.

In some embodiments of the methods and systems comprising performing mechanical separation of the microorganism, one or more reference samples can be provided by partitioning the tested sample before or after the contacting as will be understood by a skilled person.

In some embodiments, the methods and systems comprising performing mechanical separation of the microorganism can be performed on a plurality of tested samples to provide a plurality of antibiotic treated samples, wherein in each antibiotic treated sample of the plurality of antibiotic treated samples is provided by independently contacting each antibiotic tested sample for a set testing time, and in particular a testing time up to 120 minutes, and the quantitatively detecting is independently performed following the contacting at a corresponding detection time

In some embodiments, the specimen is pre-treated prior to antibiotic exposure. Pretreatment can include concentrating pathogen cells, removal of extracellular nucleic acids such as using filtration, centrifugation, or nuclease treatment, removal of interfering substances including those derived from human host such as using host cell lysis, filtration, centrifugation and nuclease treatment, and then recovery of the pathogen cells for antibiotic exposure, and any combination of these above steps.

In some embodiments, the specimen is a urine sample. In some embodiments, the antibiotic is a beta-lactam.

In some embodiments of the methods and systems herein described, the detecting can be performed in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism and in absence of sample separation.

In some of those embodiments, the quantitatively detecting is performed to detect accessible nucleic acid by performing a non-fully lytic polymerase amplification reaction such as LAMP to amplify microorganism specific target nucleic acid.

Non fully lytic or non-fully lysing polymerase amplification reactions refer to amplification reactions only partially, not fully, lysing the cell membrane and releasing a portion of intracellular contents including a portion of nucleic acids without causing cell death. Under non fully lysing polymerase amplification reaction conditions, the cell integrity is preserved to certain degree and time period to allow amplification reaction Under these conditions, most nucleic acids are protected inside cells in the control aliquot, whereas a significant portion of nucleic acid are released and start amplifying in the antibiotic treated sample.

Exemplary non fully lysing amplification reaction methods include loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase reaction (RPA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), and additional reactions identifiable by a skilled person.

In those embodiments, the quantitatively detecting is performed by measuring the TTP of the microorganism specific target nucleic acid in a treated sample following contacting with an antibiotic at exposure conditions (see Examples 25-30).

In some embodiments wherein the detecting is performed by non-fully lysing polymerase amplification, the quantitatively detecting can further be performed in a reference sample as will be understood by a skilled person upon reading of the present disclosure. In some of those embodiments, the reference sample can be a control sample wherein the non-fully lytic polymerase amplification reaction is performed in absence of contacting with the antibiotic at the exposure conditions. In some embodiments, the reference sample can be a sample treated with antibiotic as will be understood by a skilled person upon reading of the present disclosure.

In particular, in embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, the reference sample can also be a sample wherein the non-fully lytic polymerase amplification reaction is performed following contacting of the reference sample with the antibiotic at exposure conditions which differ for at least one parameter (e.g. time of contacting). In some embodiments, the reference sample can be fully lysed to detect a total nucleic acid concentration (such as total DNA, total RNA or total DNA+RNA) and the reference sample can be either treated or untreated with the antibiotics at exposure conditions that can be the same as the exposure conditions of the treated sample (if the reference sample is used to detect a total nucleic acid concentration) or different from the exposure conditions of the treated sample (if the reference sample is used to detect a reference nucleic acid concentration value other than total concentration value) as will be understood by a skilled person upon reading of the present disclosure.

In some embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, the reference sample and the treated sample can be aliquots of an original sample, as will be understood by a skilled person upon reading of the present disclosure.

In embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, and the treated and/or reference samples are provided by sub-samples, providing a reference aliquot and a treated aliquot of a sample of an isolate or specimen can comprise splitting the sample into two or more aliquots, typically of equal amounts, wherein one aliquot is treated with antibiotics and the other aliquots are independently untreated or treated with antibiotics for a certain amount of time. In some of those embodiments, the reference aliquot is used to detect a total nucleic acid concentration, and the reference aliquot can be a fully lysed aliquot created by extracting nucleic acids from an aliquot of a control sample, wherein the reference aliquot is either treated or untreated with the antibiotics.

In some embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, and wherein treated and/or reference samples are provided by sub-samples, when the reference aliquot is a reference sub-sample treated with antibiotics, the method can be performed on an antibiotic-treated sample from the contacting which is then split in aliquots following antibiotic exposure at different antibiotic contacting time. In those embodiments, using only a single aliquot of the original sample during the exposure which is further split following the contacting reduces the challenges of fluid handling and metering as will be understood by a skilled person.

In some embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, following detection of the accessible nucleic acid in the treated sample, the method can further comprise determining if the sample contains a susceptible or resistant strain based on the difference in time-to-positive between a treated samples and a reference sample. In embodiments where an original sample is split into two or more aliquots, the reference sample and treated samples are provided by one or more treated and reference aliquots as will be understood by a skilled person (see Example 25).

In embodiments of methods and systems of the present disclosure wherein detection is performed by polymerase amplification, in susceptible samples, the antibiotic compromise cell wall integrity and nucleic acids are released from compromised cells, increasing nucleic acid accessibility to the polymerase as will be understood by a skilled person. Accordingly, in embodiments wherein the detecting is performed by non-fully lysing polymerase amplification, released nucleic acid in the susceptible treated sample amplify faster than nucleic from intact cells in a control sample, resulting in a difference in time-to-positive (TTPD) and/or difference in percentage accessible nucleic acid detected that is indicative of susceptibility as would be understood by a skilled person upon reading of the present disclosure. No statistically significant difference in time-to-positive and/or percentage accessible nucleic acid between a control sample and treated sample is observed in resistant samples, as would also be understood by a skilled person upon reading of the present disclosure.

In embodiments of methods and systems of the present disclosure wherein detection is performed by polymerase amplification, the statistical significance of the TTPD can be determined by the procedure for finding a susceptible threshold and a resistant threshold as will be understood by a skilled person upon reading of the present disclosure. In general, a TTPD in the range of 1 or more minutes is statistically significant, but the specific range will depend on the characteristics of the microorganism as will be understood by a skilled person upon reading of the present disclosure (see e.g. Example 25).

In some of those embodiments susceptibility or resistance can be determined following detecting a difference in the time-to-positive between treated aliquots and treated or untreated reference aliquots. In addition or in the alternative, susceptibility or resistance can be determined following determination of a nucleic acid concentration value from the reaction time and determination of a percentage accessible nucleic acid resulting from antibiotic exposure following comparison with total nucleic acid concentrations from one or more fully lysed reference aliquots as will also be understood by a skilled person upon reading of the present disclosure.

In some embodiments of methods and systems herein described, wherein the detecting is performed by non-fully lysing polymerase amplification, and wherein the treated and reference samples comprise aliquots, the reference aliquots contain microorganism untreated with antibiotics while the treated aliquots of an equal volume as the control aliquot contains the same microorganism treated with antibiotics. The difference can be termed as TTPD_(CT), wherein C represents “control” and T represents “treated” as will be understood by a skilled person upon reading of the present disclosure (see, for example, Example 28 and FIG. 50).

In some embodiments of methods and systems herein described, wherein the detecting is performed by non-fully lysing polymerase amplification, and wherein the treated and reference samples comprise aliquots, the reference aliquots contains microorganism treated with antibiotics and fully lysed to detect a total nucleic acid concentration value. In some of those embodiments the total nucleic acid concentration value can be expressed as a total nucleic acid TTP value and a TTPD indicative of susceptibility can be provided by the difference in TTP values of the fully lysed reference aliquot and the antibiotic-treated aliquot in polymerase accessibility AST herein described. The difference can be termed as TTPD_(LT), wherein L represents “lysed” and T represents “treated” (see, for example, Example 28 and FIG. 51).

In embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, and wherein the reference sample comprise fully lysed reference sample such as fully lysed reference aliquots, the fully lysed control aliquot can be created by fully lysing a control sample using a lysis agent, wherein the control sample is either treated or untreated with the antibiotics.

In some embodiments, wherein the detecting is performed by non-fully lysing polymerase amplification, and wherein the treated and reference samples comprise aliquots, methods herein described can be performed on a treated sample which is split in a treated aliquot and a reference aliquot, without the need of a control sample and related fluid handling complexity and related incubation. In those embodiments, detection can then be performed from an aliquot of the antibiotic-treated sample using a single-step LAMP-compatible extraction buffer. Mechanical full lysis can be performed by bead beater system or a French press homogenizer.

Therefore, in those embodiments an no control sample is required, and the original sample does not have to be split prior to antibiotic exposure, thus reducing the challenges of fluid handling and metering as well the complexity of temperature control and reduces fluid handling complexity due to providing control samples and in particular aliquots which typically have same volume as treated samples or aliquots.

In some embodiments, the methods herein described wherein the detecting is performed by non-fully lysing polymerase amplification can further determine susceptibility based on the difference in the time-to-positive, i.e. TTPD_(CT) or TTPD_(LT), between the control and the treated aliquots by comparing to a predefined susceptibility threshold. If the difference in the time-to-positive is greater than the susceptibility threshold, the sample contains a susceptible isolate. If the difference in the time-to-positive is lower than the susceptibility threshold, the sample contains a resistant isolate.

In some embodiments of the methods herein described wherein the detecting is performed by non-fully lysing polymerase amplification, the susceptibility thresholds are specific to each combination of bacterial species, antibiotic, concentration of antibiotic, the specific embodiment of the methods herein described, but independent of the number of samples included in the training sets, as will be understood by a skilled person.

In some embodiments of the methods herein described wherein the detecting is performed by non-fully lysing polymerase amplification, the polymerase-accessibility ASTs described herein demonstrate good agreement with gold-standard while providing accurate susceptibility results in a reduced sample-to-answer time for a number of bacteria isolated from clinical samples. The “sample-to-answer time” refers the time required from the beginning of the sample handling till a susceptibility call is made. Sample-to-answer time directly reflects the speed of diagnostics in practice and is a major factor in how likely a diagnostic is to be adopted. In general, the shorter the sample-to-answer time, the more valuable the test is, and the more feasible for use at the Point of Care (POC).

In some embodiments of the methods herein described wherein the detecting is performed by non-fully lysing polymerase amplification, an enhancing treatment is preferably performed for treated and reference sample and in particular an enhancer preferably comprising one or more non-ionic or zwitterionic enhancers.

In some embodiments of the methods herein described wherein the detecting is performed by non-fully lysing polymerase amplification, polymerase-accessibility AST can be completed in less than 30 min. This time scale is at the same level with suggested time-frames for rapid POC diagnostics [10, 11], and measured times of patient visits [12]. The methods herein described also enables one to obtain an accurate AST result directly from a clinical sample without the need of isolating pathogens from the clinical sample. In those embodiments. the methods herein described also enable the ability to perform ASTs in parallel when testing clinical samples. The ability to run several samples in parallel demonstrates the potential to multiplex multiple antibiotics, particularly for the design of integrated devices.

In some embodiments methods and systems herein described can be performed in absence of a lysis treatment of the antibiotic treated sample targeting the microorganism; and in absence of sample separation, the methods and systems are directed to detect inaccessible nucleic acid, in those embodiments exposure conditions of the antibiotic treated and possibly the reference samples comprise a nuclease treatment.

The term “nuclease” refers to a type of enzymes capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids, such as deoxyribonucleases (DNases) and/or ribonucleases (RNases) as well as additional nucleases identifiable by a skilled person. Examples of nonspecific DNases include DNase I, RNase-free DNase I, bovine pancreatic DNase I, exonuclease I (also known as ExoI), Escherichia coli exonuclease I, Escherichia coli thermolabile exonuclease I, exonuclease III (a.k.a. ExoIII, endonuclease II, endonuclease VI), Escherichia coli exonuclease III, endonuclease IV, Escherichia coli endonuclease IV, exonuclease V (a.k.a. ExoV), Escherichia coli exonuclease V (a.k.a. RecBCD), Thermotoga maritima exonuclease V, exonuclease VII (also known as ExoVII), Escherichia coli exonuclease VII, exonuclease VIII (also known as ExoVIII), lambda phage exonuclease, micrococcal nuclease (also known as thermonuclease, MNase, staphylococcal nuclease), Benzonase nuclease (also known as Serratia marcescens endonuclease), exonuclease T (also known as RNase T), nuclease BAL-31, RecJ, RecJ_(f)™, T4 phage endonuclease II, T5 phage exonuclease, T7 phage endonuclease, T7 phage exonuclease, Endo R, S1 nuclease (also known as nuclease S1), P1 nuclease (also known as nuclease P1), mung bean nuclease, Ustilago nuclease, Neurospora nuclease, OmniCleave™ Endonuclease, Baseline-ZERO™ DNase, Plasmid-Safe™ ATP-Dependent DNase, any other DNase found naturally and manufactured recombinantly, any genetically engineered or recombinant variants of these enzymes, any fusion proteins with DNases, and any mixture of nuclease enzymes that includes an enzyme with DNase activity. Restriction enzymes from prokaryotes, CRISPR-associated proteins (including Cas9 and Cas12a), zinc-finger nucleases, and transcription activator-like effector nucleases (TALENs) are examples of sequence-specific DNases that could theoretically be used to degrade DNA non-specifically as well. Examples of RNases include RNase A, Escherichia coli RNase A, Monarch® RNase A, RNase B, RNase C, RNase H, Escherichia coli RNase H, thermostable RNase H, Hybridase™ Thermostable RNase H, human RNase H1, RNase T (a.k.a. exonuclease T),_RNase T1, RNase T2, RNase I, Escherichia coli RNase I, RNase R, RNase III, ShortCut® RNase III, Escherichia coli RNase III, human RNase3, RNase If, RNase B, RNase HII, micrococcal nuclease (a.k.a. thermonuclease, MNase, staphylococcal nuclease), S1 nuclease OmniCleave™ Endonuclease, Terminator™ 5′-Phosphate-Dependent Exonuclease, RNase L, RNase P, any other RNase found naturally and manufactured recombinantly, any genetically engineered or recombinant variants of these enzymes, any fusion proteins with RNases, and any mixture of nuclease enzymes that includes an enzyme with RNase activity.

Methods of the present disclosure can be performed with a corresponding system comprising at least one probe specific for a nucleic acid of the target microorganism and reagents for detecting the at least one probe. The at least one probe and reagents are included in the system for simultaneous combined or sequential use in any one of the methods of the present disclosure.

In some embodiments of the system herein described the system comprises primers configured to specifically hybridize with a sequence of nucleic acid from the target organism.

In some embodiments, the systems of the disclosure to be used in connection with methods herein described can further comprise an antibiotic formulated for administration to a sample in combination with the at least one probe.

In some embodiments, the systems of the disclosure, the system further comprises an antibiotic formulated for administration to an individual in an effective amount to treat a microorganism infection in the individual.

In some embodiments, the systems of the disclosure to be used in connection with methods herein described, the reagents comprise DNA extraction, RNA extraction kit and amplification mix. The system can also include one or more antibiotics and/or exposure media with or without the antibiotics. The system can also include reagents required for preparing the sample, such as one or more of buffers e.g. lysis, stabilization, binding, elution buffers for sample preparation, enzyme for removal of DNA e.g. DNase I, and solid phase extraction material for sample preparation, reagents required for quantitative detection such as intercalating dye, reverse-transcription enzyme, polymerase enzyme, nuclease enzyme (e.g. restriction enzymes; CRISPR-associated protein-9 nuclease; CRISPR-associated nucleases as described herein) and reaction buffer. Sample preparation materials and reagents may include reagents for preparation of RNA and DNA from samples, including commercially available reagents for example from Zymo Research, Qiagen or other sample preparations identifiable by a skilled person. The system can also include means for performing DNA or RNA quantification such as one or more of: container to define reaction volume, droplet generator for digital quantification, chip for digital detection, chip or device for multiplexed nucleic acid quantification or semiquantification, and optionally equipment for temperature control and detection, including optical detection, fluorescent detection, electrochemical detection.

In some embodiments, the system can comprise a device combining all aspects required for an antibiotic susceptibility test.

The systems herein disclosed can be provided in the form of kits of parts. In kit of parts for performing any one of the methods herein described, the probes and the reagents for the related detection can be included in the kit alone or in the presence of one or more antibiotic as well as any one of the RNA markers, corresponding cDNA and/or probes for one or more reference RNAs and/or corresponding cDNAs. In kit of parts for the treatment of an individual the probes and reagents for the related detection can be comprised together with the antibiotic formulated for administration to the individual as well as additional components identifiable by a skilled person.

In a kit of parts, the probes and the reagents for the related detection, antibiotics, RNA markers, and/or reference RNA and additional reagents identifiable by a skilled person are comprised in the kit independently possibly included in a composition together with suitable vehicle carrier or auxiliary agents. For example, one or more probes can be included in one or more compositions together with reagents for detection also in one or more suitable compositions.

Additional components can include labeled polynucleotides, labeled antibodies, labels, microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.

The terms “label” and “labeled molecule” as used herein refer to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemoluminescence, production of a compound in outcome of an enzymatic reaction and the like.

In embodiments herein described, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here disclosed. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes, CD-ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).

Further details concerning the identification of the suitable carrier agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.

EXAMPLES

The methods of the disclosure and related composition, and systems herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary methods and protocols for performing methods directed to detect accessible or inaccessible nucleic acid. A person skilled in the art will appreciate the applicability and the necessary modifications to adapt the features described in detail in the present section, to additional methods and related compositions and systems according to embodiments of the present disclosure.

The following general materials and methods were used unless specified.

Clinical isolates referenced, minimum inhibitory concentrations (MICs), and categorical susceptibility based on CLSI breakpoints The below list of organisms are examples of clinically-relevant susceptible and resistant Enterobacteriaceae clinical isolates. Minimum inhibitory concentrations (MICs) are provided in μg/mL for the antibiotics listed. Any number with an inequality sign ≤, or > indicates that an endpoint measurement was not made.

Antibiotic MICs ID Organism AMP CRO ETP Ec_1 Escherichia coli   4 * <=1 * <=.5 * Ec_2 Escherichia coli  <=2 * <=1 * <=.5 * Ec_3 Escherichia coli  <=2 * <=1 * <=.5 * Ec_4 Escherichia coli  <=2 * <=1 * <=.5 * Ec_38 Escherichia coli ND >32 # >4 #   Ec_39 Escherichia coli >=32 # >32 # 2 #  Ec_40 Escherichia coli >=32 # >=64 #  2 #  Ec_41 Escherichia coli ND >32 # 2 #  Kp_1 Klebsiella pneumoniae ND <=1 * <=.5 * Kp_2 Klebsiella pneumoniae ND <=1 * <=.5 * Kp_7 Klebsiella pneumoniae  >32 # >32 # >8 #   Kp_8 Klebsiella pneumoniae  >32 # >32   >8 #   * the organism is susceptible (also referred to herein as “S”), # the organism is resistant (also referred to herein as “R”) ND = not determined

The below list of organisms are examples of clinically-relevant susceptible and resistant Neisseria gonorrhoeae clinical isolates. Minimum inhibitory concentrations (MICs) are provided in μg/mL for the antibiotics listed. Any number with an inequality sign ≤, or > indicates that an endpoint measurement was not made.

Antibiotic MICs ID Organism CIP CRO CFM PEN ng_1 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 * 0.250 ng_2 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 * 0.250 ng_3 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 * 0.250 ng_4 Neisseria gonorrhoeae 16.000 #  0.030 *  0.060 * 0.500 ng_5 Neisseria gonorrhoeae <0.015 *  0.015 *  0.030 * 1.000 ng_6 Neisseria gonorrhoeae  4.000 # <0.008 *  0.015 * 0.250 ng_12 Neisseria gonorrhoeae  2.000 #  0.015 *  0.030 * 0.500 ng_15 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 *   0.015 * ng_16 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 *   0.015 * ng_17 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 *   0.060 * ng_18 Neisseria gonorrhoeae 16.000 #  0.030 *  0.060 *   2.000 # ng_19 Neisseria gonorrhoeae  4.000 #  0.015 *  0.030 *   2.000 # ng_20 Neisseria gonorrhoeae <0.015 * <0.008 * <0.015 *   0.060 * ng_30 Neisseria gonorrhoeae  0.015 *  0.500 #  1.000 # 1.000 ng_41 Neisseria gonorrhoeae 16.000 #  0.125 *  0.250 *   2.000 # ng_44 Neisseria gonorrhoeae  4.000 # <0.008 * <0.015 *   8.000 # * the organism is susceptible (also referred to herein as “S”), # the organism is resistant (also referred to herein as “R”) ND = not determined

MHB Media Preparation: Mueller Hinton Broth (MHB) prepared from BBL Mueller Hinton II Broth Cation Adjusted (BBL cat no. 212322) according to manufacturer instructions (autoclaved to sterilize)

HFB Media Preparation: Hardy fastidious broth (HFB) purchased from Hardy Diagnostics (Hardy cat no. K31) and used as received (stored at 4° C.)

BHI Media Preparation Brain-heart Infusion Media (BHI) prepared from BD Bacto Brain Heart Infusion (BD cat no. 237500) according to instructions (autoclaved to sterilize)

GWM Media Preparation M199 cell culture medium was prepared by dissolving M199 salts (Sigma cat no. M3769, lot no. SLBW4106) in 1 L milliQ H2O. Graver-Wade salt additions to 667 mL M199 cell culture medium (final volume 1.0 L) are shown below. All concentrations based on volumes and reported concentration in Wade et al. 2007 paper. Final media is filter-sterilized with a 0.2 μm filter and stored at 4° C.

Volume Component (stock concentration) (final concentration) Glucose (1000 mM) 3.7 mL (37 mM) Ammonium bicarbonate (1000 mM) 1.7 mL (17 mM) Sodium acetate (1000 mM) 0.49 mL (4.9 mM) L-glutamine (50 mM) 6.8 mL (3.4 mM) Spermidine (500 mM) 0.18 mL (0.92 mM) L-arginine (100 mM) 0.38 mL (0.38 mM) Hypoxanthine (100 mM) 0.25 mL (0.25 mM) Uracil (850 mM) 0.35 mL (0.30 mM) Oxaloacetate (100 mM) 0.25 mL (0.25 mM) Thiamine hydrochloride (100 mM) 0.10 mL (0.10 mM) L-ornithine (100 mM) 0.04 mL (0.04 mM) Nicotinamide adenine dinucleotide, 0.02 mL (0.01 mM) NAD+ (50 mM) DL-lactate (60% w/w, ~7500 mM) 2.5 mL (~13 mM) NF-H2O 316.24 mL

Preparation and storage of antibiotic stocks: All antibiotic (ABX) stocks are prepared from solids of the listed antibiotic or solid salts of the listed antibiotic. ABX stocks are prepared such that the final concentration of antibiotic (not including counterion salts) is as listed. ABX stocks were prepared at 1 mg/mL in either nuclease-free H₂O (NF—H2O) unless listed otherwise. 50-100 uL aliquots of ABX stocks were stored frozen at −80 celsius. Aliquots are thawed only once on day of experiment before use, and used to prepare listed working stock.

NG Stock and Resuspension: the following procedure is carried out:

-   -   1. Isolates are plated from glycerol stocks onto BBL ChocII         Agar—Pre-poured plates (Cat no. 221267) and grown overnight in a         37° C., 5% CO₂ incubator. (Referred to as “pass 0” or “p0”         plates).     -   2. Isolates are passed by streaking a colony or a clump of         colonies on a new, pre-warmed BBL Chocolate II Agar plate. The         passage number is increased each time the plates are streaked         this way; cells from a p0 plate streaked onto a fresh BBL         Chocolate II Agar plate are now labeled p1 cells.         -   a. Note: cultures started for resuspensions for experiment             prep use pass 1 (p1), pass 2 (p2), or pass 3 (p3) plates.     -   3. Prewarm media before adding cells for at least 30-60 minutes         in a 37° C., 5% CO₂ incubator on a shaking block (500-1000 rpm)         before adding cells (step 4).         -   a. Media is either MHB with an addition of NaHCO₃ to a final             concentration of 5 mM or GWM. Each cell pellet is             resuspended in 2 mL of pre-warmed media.     -   4. Resuspend cell pellets. Scrape cell pellet from the chocolate         agar plate with inoculating loop and twirl in 2 mL pre-warmed         media. incubate on shaker (1000 rpm) in 37° C., 5% CO₂ incubator         -   a. Break apart the cell pellet by pipetting the cell pellet             up and down with a P1000 until large pieces are no longer             seen.         -   b. Resuspension time can vary from 10 minutes-8 hours,             depending on the objective of the experiment.

EC, KP, Stock and Resuspension All Ec and Kp stocks were handled in the same fashion as Ng stocks, except plates used to grow Ec and Kp were TSA II plates with 5% sheep's blood. Media used for resuspension was typically BD brain heart infusion media or mueller hinton broth unless listed otherwise. Media used for incubation was typically MHB unless listed otherwise.

DEB Extraction: the extraction can be performed with the following materials: Lucigen QuickExtract DNA Extraction Soln. 1.0. Cat #QE09050, and according to the following procedure:

-   -   1. In the BioSafety Cabinet (BSC), dilute sample 1/10 into         Lucigen DNA Extraction Buffer (DEB). Such as 10 μL from exposure         tube into 90 μL DEB, in a 1.5 mL Eppendorf tube or a PCR tube.         -   a. Gently close tubes         -   b. Vortex for 2-3 seconds         -   c. Quickly centrifuge tubes to remove the droplets from the             caps         -   d. Spray gloves and tubes with 70% ethanol before moving to             the thermocycler from the BSC (If using Eppendorf tubes,             move to a glass bead bath set to 65° C.)     -   2. On thermocylcer or heat blocks:         -   a. Heat to 65° C. for 6 minutes         -   b. Heat to 98° C. for 4 minutes         -   c. Hold at 12° C. (PCR tubes on thermocylcer) or move to ice             bath (Eppendorf tubes) to cool             The heating can be done on the BioRad thermocylcer in PCR             tubes, or in sequential heating steps on heat-blocks in             Eppendorf tubes.

Zymo Column Extraction (RNA): the extraction can be performed with the following materials: bacterial suspension to extract RNA and DNA (referred to as “sample”), Zymo Research DNA/RNA Shield, Zymo Research Viral DNA/RNA kit, and according to the following procedure:

-   -   1. Add 1 volume of DNA/RNA Shield to 1 volume sample (50 μL:50         μL)     -   2. Add 2 volumes Viral DNA/RNA Buffer to sample and mix (200 μL         buffer+100 μL sample+shield)     -   3. Transfer sample to Zymo Spin column in a collection tube and         centrifuge for 1-2 minutes. Discard flow-through     -   4. Add 500 μL viral wash buffer to column and centrifuge for 2         minutes.     -   5. Transfer column to a DNase/RNase-Free Tube.     -   6. Elute: Add 50-100 μL Nuclease-Free Water and centrifuge for         30 seconds.

qPCR: qPCR mixes were prepared according to the table below.

qPCR Component (stock concentration) Volume (final concentration) Initial Melt (1X) BioRad SsoFast EvaGreen Supermix 5.0 uL (1X) 95° C. 3.0 min (2X) PCR primer mix (10 uM each) 0.5 uL (0.5 uM each) Cycle (40X) NF-H20 3.5 uL 95° C. 10 sec Template 1.0 uL 60° C. 10 sec 72° C. 15 sec Melt Curve (1X) 95° C. 15 sec 50° C. 15 sec 50-95° C. cont. read

PCR primers used for detection and quantification of Ec and Kp consisted of forward and reverse primers with sequence TGCCGTAACTTCGGGAGAAGGC (SEQ ID NO: 1) and TCAAGGCTCAATGTTCAGTGTC (SEQ ID NO: 3) respectively, and were obtained from Matsuda et al. 2007 [24]. PCR primers used for detection and quantification of Ng consisted of forward and reverse primers with sequence ACTGCGTTCTGAACTGGGTG (SEQ ID NO: 4) and GGCGGTCAATTTCACGCG (SEQ ID NO: 2) respectively, and were obtained from Lee et al. 2007 [25].

The thermocycling can be performed according to the following procedure

-   -   1. If possible, run triplicate wells for each nucleic acid         sample in 10 μL volumes according to the table below of the qPCR         master mix:

qPCR Master Mix (V_(f) = 10 μL) Reagent Volume BioRad sso fast (contains EvaGreen) 2X 5.0 μL (1X) Primer Mix (10 μM forward and reverse) 0.5 μL (0.5 μM) Template: Extracted DNA 1.0 μL Nuclease-Free Water 3.5 μL

-   -   2. Vortex and centrifuge each of the PCR tubes with three         volumes of the Master Mix prior to aliquoting into the Roche         Light Cycler 96 plate.     -   3. With the PCR plate on a −20° C. cold block, pipette 9.5 μL         into each well of the PCR well plate.     -   4. Seal the well plate with film.     -   5. Centrifuge the well plate     -   6. Place the well plate back to the −20° C. ice block until it         is placed on the machine. Run the protocol as follows:     -   7. An example of PCR settings are reported in the table below

Initial Melt 95° C. 3:00 35X 95° C. 0:15 62° C. 0:15 72° C. 0:20 Melt Curve 1X 95° C. 0:15 50° C. 0:15 95° C.

-   -   8. Export the data from the Roche LightCylcer 96 software to a         .csv or .xlsx file. Perform the analysis on the Cq data         (plotting the raw Cq data and processing the Cqs to get a C:T         ratio)

qRT-PCR: the qRT PCR can be performed with the mix and conditions summarized in the tables below.

RT-qPCR mix (10 μL) For -RT samples, WarmStart RTx and RiboGuard volume was replaced with NF-water. Component (stock concentration) Volume (final concentration) 2X BioRad ssoFast EvaGreen Supermix 5.0 μL (1X) WarmStart RTx (15,000 U/mL) 0.1 μL (150 U/mL) RiboGuard (40,000 U/mL) 0.2 μL (800 U/mL) Primer Mix (10 μM each) 0.5 μL (0.5 μM each) Template 1.0 μL NF water 3.2 μL

RT-qPCR rxn conditions RT/Initial Melt (1X) 55° C. 10 min 95° C. 3 min Cycle (30X) 95° C. 15 s 62° C. 15 s 72° C. 15 s Melt Curve (1X) 95° C. 15 s 50° C. 15 s 95° C. 5 s

ddPCR: ddPCR can be performed with the material and conditions summarized in the table below

Component (stock concentration) Volume (final concentration) RT (1X) BioRad Droplet Generation Mix for 5.0 uL (1X) 55° C. 10.0 min EvaGreen (2X) Primer mix (10 uM each) 0.5 uL (0.5 uM each) Initial Melt (1X) NF-H2O 3.5 uL 95° C. 5.0 min Template 1.0 uL Cycle (40X) 95° C. 30 sec 60° C. 30 sec 68° C. 30 sec Dye Stabilization (1X)  4° C. 5.0 min 90° C. 5.0 min 12° C. Hold

ddRT-PCR the ddRT PCR can be performed with the mix and conditions summarized in the tables below.

RT-ddPCR mix (10 μL). For -RT samples, WarmStart RTx and RiboGuard volume was replaced with NF-water. Volume (final Component (stock concentration) concentration) 2X BioRad Droplet Generation Mix for 5.0 μL (1X) EvaGreen WarmStart RTx (15,000 U/mL) 0.1 μL (150 U/mL) RiboGuard (40,000 U/mL) 0.2 μL (800 U/mL) Primer Mix (10 μM each) 0.5 μL (0.5 μM each) Template 1.0 μL NF water 3.2 μL

RT-ddPCR rxn conditions 2° C./s ramp on all cycling steps. RT/Initial Melt (1X) 55° C. 10 min 95° C. 3 min Cycle (30X) 95° C. 15 s 60° C. 15 s 68° C. 15 s Dye Stabilization (1X)  4° C. 5 min 90° C. 5 min 12° C. hold

qLAMP without enhancer present in amplification mix; the qLAMP can be performed with the material and conditions summarized in the table below

LAMP Component (stock concentration) Cycle (120X) IsoAmp Butter II (10X, (—) Tween 20) 1.0 uL (1X) 70° C. 10 sec MgSO4 (100 mM) 0.3 uL (5 mM) Melt Curve (1X) KCI (1.0 M) 1.5 uL (150 mM) 95° C. 15 sec dNTPs (10 mM each) 1.4 uL (1.4 mM each) 50° C. 15 sec Primer mix (20 X) 0.5 uL (1X) 50-95° C. cont. read Bst 3.0 (8000 U/mL) 0.4 uL (320 U/mL) Syto-9 (50 uM) 0.4 uL (2 uM) Template 1.0 uL NF-H2O 3.5 uL

wherein the Iso Amp Buffer II (10×, (−) Tween 20)=200 mM Tris-HCl, 20 mM MgSO4, 100 mM (NH4)2SO4, pH 8.8. Primers for detection and quantification of Ec and Kp was same as that listed in Schoepp et al. 2017 [26].qLAMP with enhancer present in amplification mix: can be performed with the material and conditions summarized in the table below

Component (stock concentration) Volume (final concentration) Cycle (120X) NEB IsoAmp Buffer II 1.0 uL (1X) 70° C. 10 sec MgSO4 (100 mM) 0.3 uL (5 mM) Melt Curve (1X) dNTPs (10 mM each) 1.4 uL (1.4 mM each) 95° C. 15 sec Primer mix (20X) 0.5 uL (1X) 50° C. 15 sec Bst 3.0 (8000 U/mL) 0.4 uL (320 U/mL) 50-95° C. cont. read Syto-9 (50 uM) 0.4 uL (2 uM) Template 1.0 uL NF-H2O 5.0 uL wherein the NEB Iso Amp Buffer II=200 mM Tris-HCl, 20 mM MgSO4, 100 mM (NH4)2SO4, pH 8.8. Primers for detection and quantification of Ec and Kp was same as that listed in Schoepp et al. 2017 [26].

The following general materials and methods were used in Examples 24 to 30, unless specified.

Isolates, growth conditions, and antibiotic exposure conditions. 25 de-identified clinical isolates were obtained from the UCLA Clinical Microbiology Laboratory (CML) and the CDC's Enterobacteriaceae Carbapenem Breakpoint panel[3]. In the case of isolates obtained from the UCLA CML, MICs were determined as described previously[4]. Genotypic testing of the two Ec and two Kp isolates selected for their lack of known β-lactamase genes was performed by UCLA CML using a previously published assay[5] and separately at the Keck School of Medicine of USC using the FDA-approved Cepheid Xpert® Carba-R test. Whole genome sequencing of the single Ebs isolate selected for its lack of known resistance genes was performed by the CDC[3]. All isolates were stored as glycerol stocks at −80° C. Glycerol stocks were streaked onto Trypticase Soy Agar with 5% sheep's blood (Becton Dickinson, Franklin Lakes, N.J., USA) and grown overnight at 37° C. Prior to experiments, a small clump of cells was resuspended in 2 mL Brain Heart Infusion Broth (BHI, Becton Dickinson) at 37° C.+5% CO₂ with 500 rpm shaking for 2 to 4 h until visibly turbid. OD₆₀₀ of the cultures was then measured, and working cultures prepared at an OD₆₀₀ of 0.01-0.07 and grown for 50-145 min at 37° C.+5% CO₂ with 500 rpm. Working cultures were then diluted 10× into control and treated aliquots for antibiotic exposure. For validation experiments, antibiotic exposure was performed in 100 μL volumes consisting of 80 Mueller Hinton II Broth (Becton Dickinson), 5 μL nuclease-free H₂O (NF—H₂O), 5 μL 20× antibiotic stock solution, and 10 μL of working culture. In control aliquots, antibiotic stock solution was replaced with NF-H₂O. For filtration experiments, antibiotic exposure was performed in 100 μL volumes consisting of 65 μL Mueller Hinton II Broth (Becton Dickinson), 21 nuclease-free H₂O (NF—H₂O), 4 μL 25× antibiotic stock solution, and 10 μL of working culture. In control aliquots, antibiotic stock solution was replaced with NF—H₂O.

Antibiotic stocks. Ceftriaxone disodium salt hemi(heptahydrate) (Sigma, St. Louis, Mo., USA), ertapenem sodium salt (Research Products International, VENDOR), and meropenem trihydrate (TCI, Portland, Oreg., USA) were used to create antibiotic stock solutions. All antibiotic stock solutions were prepared at 1.0 mg/mL antibiotic in nuclease-free H₂O (NF—H₂O) based on manufacturer reported purity, aliquoted, and stored at −80° C. Aliquots were only thawed and used once on the days of experiments.

Comparison of amplification methods. In order to compare amplification using LAMP and PCR, E. coli isolates were exposed to 0.5 μg/mL ertapenem for 15 min. Samples were then transferred directly into either PCR or LAMP mix on ice. Amplification was started immediately. Quantitative PCR (qPCR) was performed on a Roche LightCycler 96 using SsoFast EvaGreen Supermix (BioRad, Hercules, Calif., USA). 10 μL reactions were used. 10% of the final reaction volume was template. Published primers were used[6] at a final concentration of 500 nM. Cycling conditions consisted of 3.0 min at 95° C., followed by 35 cycles of 95° C. for 10 sec, 60° C. for 10 sec, and 72° C. for 15 sec. Fluorescence was measured using the SYBR Green channel after each 72° C. extension step. LAMP was performed on a BioRad CFX96 using the following conditions: 10 μL reaction volume containing 1× Isothermal Reaction Buffer II (NEB), 5 mM MgSO4 (NEB), 1.4 mM dNTPs (NEB), 320 U/mL Bst 3.0 (NEB), and 2 μM Syto-9 (Thermo Fisher). 10% of the reaction volume was template. Primer sequences and concentrations have been described previously[2]. Cycling conditions consisted of 2.0 min at 12° C. (while lid was heating), followed by 120 cycles of 70° C. for 10 sec. Fluorescence was measured using the SYBR Green channel every 10 sec (after each cycle).

Filtration experiments. Filtration experiments were performed using E. coli isolates exposed to 0.5 μg/mL ertapenem for 15 min. Immediately following exposure, cultures were passed through 0.22 μm, 1.5 mL cellulose acetate centrifuge tube filters (Corning Costar Spin-X, Corning, N.Y.) by adding 50 μL of sample to the filter and centrifuging for 4 minutes at 1000 rcf. DNA was extracted from both the feed and filtrate using QuickExtract DNA Extraction Solution (Lucigen, Middleton, Wis., USA). Samples were diluted 10× into extraction buffer and extracted according to manufacturer instructions. The concentration of the single copy E. coli uidA gene was then quantified in the feed and filtrate extractions. The percentage of E. coli DNA in the filtrate was calculated as the filtrate concentration divided by the feed concentration. Droplet digital PCR (ddPCR) was performed using QX200 ddPCR Supermix for EvaGreen (BioRad). 10% of the final reaction volume was template. Published primers targeting the uidA gene in E. coli were used[7] at a final concentration of 500 nM. Cycling conditions consisted of 5.0 min at 95° C., followed by 40 cycles of 95° C. for 30 sec, 60° C. for 30 sec, and 72° C. for 30 sec., with final dye stabilization steps of 4° C. for 5.0 min followed by 90° C. for 5.0 min.

pol-aAST Validation. For pol-aAST validation experiments, E. coli and Enterobacter spp. isolates were exposed to either 2.0 μg/mL ceftriaxone (CRO), 0.5 μg/mL ertapenem (ETP), or 1.0 μg/mL meropenem (MEM). K. pneumoniae isolates were exposed to either 2.0 μg/mL CRO, 1.0 μg/mL ETP, or 1.0 μg/mL MEM. Some isolates were run multiple times on different days. If this was the case, the average TTPD_(CT) and TTPD_(LT) are reported for that isolate. All isolates were exposed to ABXs for 15 min in 100 μL reaction volumes in 200 μL PCR tube strips. After 15 minutes of ABX exposure, 10 μL of samples were transferred as template to LAMP reaction mix (as described above) on ice. Amplification was immediately started.

Timed sample-to-answer using contrived urine samples. Timed sample to answer experiments were performed in the same fashion as pol-aAST validation experiments, except with the following modifications. Following initial growth and measurement of OD, isolates were resuspended in fresh, never-frozen, pooled human urine from healthy donors (Lee BioSciences). Additionally, a timer was started as soon as samples were added to the ABX exposure conditions. Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) isolates were exposed to 0.5 and 1.0 μg/mL ETP (respectively) for 13 min. 13 min was chosen to ensure all handling steps could be completed within the first 15 min of the assay. Amplification was performed until all reactions reached a fluorescence value of 1000 relative fluorescent units (RFU) or greater. Amplification was then stopped, and TTP values copied into a spreadsheet pre-populated with formulas to automatically output susceptibility calls. The timer was stopped once a susceptibility call had been determined.

Testing of pol-aAST with clinical samples. UCLA CML performed urinalysis, confirmation of urinary tract infection, pathogen isolation and identification, and subsequent gold-standard AST using broth microdilution. Gold-standard AST results were sent to Caltech researchers on the same day samples were received. Enterobacteriaceae-positive samples were shipped at ambient temperature to Caltech in BD Vacutainer Plus C&S preservative tubes (Becton Dickinson, Cat. 364951) containing a boric acid preservative. The pol-aAST experiments were performed directly on these samples within 3-5 d of their collection at UCLA. Urine samples were first warmed up to 37° C. without shaking for 30 min, to approximate temperature of freshly collected urine. Then, 30 μL of urine was diluted into 70 μL of Cation-adjusted Mueller Hinton II Broth (MHB, BD) containing 0.1% Tween-20 (Teknova) and placed at 37° C. with shaking at 750 rpm for 3 min. Samples were then centrifuged at 5000 rcf for 2 min. The supernatant was removed, and the sample resuspended in 100 μL of MHB. Samples were then incubated for 30 min at 37° C. with 750 rpm shaking. ABX exposure was performed in a final volume of 100 after transfer of 20 μL of incubated sample to 80 μL of the exposure condition: 75 μL of MHB and 5 μL of 20× ABX stock solution in NF—H2O for treated aliquots, or 75 μL of MHB with 5 μL of NF—H2O alone for control aliquots. For measurement of ETP susceptibility, the exposure condition contained a final concentration of 1 μg/mL of ETP. Aliquots were incubated at 37° C. with shaking for 20 min. For measurement of AMP susceptibility, the ABX-exposure condition contained a final concentration of 16 μg/mL of AMP, and aliquots were incubated at 37° C. with shaking for 45 min. The control and treated aliquots were subjected to a set of dilutions to account for variable bacterial load of the samples and resolution within the working range of the LAMP reaction. Following dilution, 1 μL of the control and treated aliquots was added to each LAMP reaction well. There were three technical replicates (3 LAMP reaction wells) for each condition (control and treated). We measured the time-to-positive (TTP) for the reactions at each dilution, and then selected the dilution that yielded a control TTP value later than 4.7 min. The TTP results from this dilution were used to calculate TTPD_(CT) (and determine susceptibility). Samples with a TTPD_(CT)>0.25 min were considered susceptible, while samples with TTPD_(CT)z≤ to 0.25 min were considered resistant. The susceptibility determination of the pol-aAST method was then compared to the gold-standard culture results obtained by the UCLA CML to measure assay performance.

Statistical analysis. Significance referenced in the text for Pola-FIG. 2 were calculated using GraphPad Prism 8.0 software from an unpaired, two-tailed t-test comparing the averages of three replicate Cq values of each control sample to each treated sample. A significance value of 0.02 was used for statistical significance. All percent release values (FIG. 49) and TTPD values (FIGS. 50-52) were calculated using Microsoft Excel. Data were plotted using GraphPad Prism 8.0 software. Thresholds for determining susceptibility in TTPD_(CT) and TTPD_(LT) plots were set halfway between the lowest S and highest R values for each organism/ABX combination. For preliminary tests with clinical samples, we defined a TTPD_(CT) of above 0.25 min for a susceptible determination; this value would be further defined in a subsequent larger-scale clinical trial.

The following materials and methods were used for Example 31 to 36 unless otherwise specified.

Isolates and agar-dilution MIC testing. Isolates were provided by the University of Washington Neisseria Reference Laboratory and the Centers for Disease Control (CDC) Antibiotic Resistance Isolate Bank Neisseria gonorrhoeae panel (S1Nuca-Table). MICs of the CDC AR Isolate Bank are reported by the CDC [10], and MIC of all other isolates were determined by agar dilution according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [11].

Reagents and culture media. BD BBL Chocolate II Agar prepared plated media (GC II Agar, with Hemoglobin and BD IsoVitaleX) was purchased from VWR International LLC (VWR, Radnor, Pa., USA). Graver-Wade Medium (GWM) was prepared as described previously [12]. Cation-adjusted Mueller Hinton II Broth (MHB) (BD, Franklin Lakes, N.J., USA) was prepared according to manufacturer instructions. All sodium bicarbonate (NaHCO₃) (Sigma, St. Louis, Mo., USA) and calcium chloride (CaCl₂) (Fisher Scientific, Hampton, N.H., USA) stocks were dissolved in nuclease-free water (NF—H₂O) and sterilized using 0.2-μm filters. DNase I (2000 U/mL) was obtained from New England Biolabs (NEB; Ipswich, Mass., USA). Normal urine from pooled human donors was purchased from Lee Biosolutions (Maryland Heights, Mo., USA) and filtered through 0.2-μM filters before use.

Antibiotic stocks were prepared and stored as single-use aliquots at −80° C. Aliquots were thawed once and diluted in NF—H₂O before use. PEN (1 mg/mL) was prepared from penicillin G sodium salt (Sigma, St. Louis, Mo., USA) in NF-H₂O. CRO (1 mg/mL) was prepared from ceftriaxone disodium salt hemi(heptahydrate) (Sigma) in NF-H₂O. CFM (5 mg/mL) was prepared from cefixime trihydrate (Sigma) in DMSO.

Unless otherwise noted, enhancer stock solutions were prepared in NF—H₂O and stored at room temperature. Tris buffer (500 mM; pH 8.5 at 37° C.) was prepared according to the Sigma buffer reference tables [13] using 0.2-μm filter sterilized stocks of 1 M Tris-HCl (Sigma) and 1 M Tris base (Fisher Scientific) prepared in milliQ H₂O. TNP HLB 13.1 (100 mM) was prepared by mixing 334 μL 100 mM Tergitol NP-9 (Sigma)+666 μL 100 mM Tergitol NP-10 (Sigma). CHAPS (200 mM) was prepared from CHAPS solid powder (Sigma). 0.1% sodium dodecyl sulfate (SDS) was prepared by diluting 10% SDS (Invitrogen, Carlsbad, Calif., USA). BAC (10%) was prepared from benzalkonium chloride solid powder (MP Biomedicals, Santa Ana, Calif., USA).

Nucleic acid quantification. Quantitative PCR (qPCR) was performed using ssoFast EvaGreen Supermix (BioRad, Hercules, Calif., USA) in 10 μL reactions with 500 nM primers targeting the Ng 16S rRNA gene [14]. DNA template composed 10% of the reaction volume. Cycling conditions consisted of 3.0 min at 95° C., followed by 35 cycles of 15 sec at 95° C., 15 sec at 62° C., and 20 sec at 72° C. All qPCR was performed on either a Roche LightCycler 96 or BioRad CFX96 instrument. The Cq values obtained from qPCR are used to compute the percentage accessibility and percentage lysis as described in the equations below. Any negative percentages were set to 0 for plotting.

$\begin{matrix} {{\%\mspace{14mu}{Accessibility}\mspace{14mu}\left( {{control}\mspace{14mu}{and}\mspace{14mu}{treated}} \right)} = {\left( {1 - 2^{({{cq_{cont{rol}}} - {cq_{treated}}})}} \right) \times 100}} & (1) \\ {\mspace{79mu}{{{PCR}\mspace{14mu}{error}\mspace{14mu}\%\mspace{14mu}{Accessibility}} = {\sqrt{\left( \frac{\sigma_{C}}{\mu_{C}} \right)^{2} + \left( \frac{\sigma_{T}}{\mu_{T}} \right)^{2}} \times 100}}} & (2) \end{matrix}$

-   -   C=linearized control Cqs=2^(Cq) ^(control)     -   T=linearized treated Cqs=2^(Cq) ^(treated)     -   σ=standard deviation     -   μ=mean

$\begin{matrix} {{\%\mspace{14mu}{Lysis}\mspace{14mu}\left( {{no}\mspace{14mu}{enhancer}\mspace{14mu}{and}\mspace{14mu}{enhancer}} \right)} = {\left( {1 - 2^{({{cq_{{no}\mspace{14mu}{enhancer}}} - {cq_{enhancer}}})}} \right) \times 100}} & (3) \\ {\mspace{79mu}{{{PCR}\mspace{14mu}{error}\mspace{14mu}\%\mspace{14mu}{Lysis}} = {\sqrt{\left( \frac{\sigma_{N}}{\mu_{N}} \right)^{2} + \left( \frac{\sigma_{E}}{\mu_{E}} \right)^{2}} \times 100}}} & (4) \end{matrix}$

-   -   N=linearized no enhancer Cqs=2^(cq) ^(no enhancer)     -   E=linearized enhancer Cqs=2^(Cq) ^(enhancer)     -   σ=standard deviation     -   μ=mean

Droplet digital PCR (ddPCR) was performed using QX200 ddPCR Supermix for EvaGreen (BioRad, Hercules, Calif., USA) with the same primers and primer concentrations used in qPCR. DNA template composed 10% of the reaction volume. Cycling conditions consisted of 5.0 min at 95° C., followed by 40 cycles of 30 sec at 95° C., 30 sec at 60° C., and 30 sec at 72° C., followed by a droplet stabilization step of 4° C. for 5 min, and 95° C. for 5 min. Calculations of percentage accessibility and percentage lysis for ddPCR are given the equations below, where □ represents template concentration in copies/μL. The template concentrations are used to compute percentage accessibility and percentage lysis as described in the equations below. Any negative percentages were set to 0 for all analyses.

$\begin{matrix} {\mspace{79mu}{{\%\mspace{14mu}{Accessibility}\mspace{14mu}\left( {{control}\mspace{14mu}{and}\mspace{14mu}{treated}} \right)} = {\left( {1 - \left( \frac{\lambda_{treated}}{\lambda_{control}} \right)} \right) \times 100}}} & (5) \\ {{\%\mspace{14mu}{Lysis}\mspace{14mu}\left( {{no}\mspace{14mu}{enhancer}\mspace{14mu}{and}\mspace{14mu}{enhancer}} \right)} = {\left( {1 - \left( \frac{\lambda_{enhancer}}{\lambda_{{no}\mspace{14mu}{enhancer}}} \right)} \right) \times 100}} & (6) \end{matrix}$

A dLAMP assay was performed using a previously published system [15]. The dLAMP mix consisted of 1 μL NEB Isothermal Amplification Buffer (200 mM Tris-HCl, 20 mM MgSO4, 500 mM KCl, 100 mM (NH₄)₂SO₄, 1% Tween 20, pH 8.8), 0.6 μL MgSO4, 0.5 μL BSA (20 mg/mL), 0.4 μL Syto-9 (50 μM, prepared within two weeks of use), 1.4 μL dNTPs (10 mM each), 0.5 μL 20× primer mix, 0.4 μL NEB Bst 2.0 WarmStart, 0.2 μL Ambion RNase cocktail, 4.0 μL NF-H₂O, and 1 μL of template. Primers were designed to target the Ng 16S gene, and screened as described previously [9]. Primer sequences used are as follows, with the final concentration in the amplification mix in parentheses: GCGGTGGATGATGTGGATT (SEQ ID NO: 5) (forward outer primer, 0.2 μM), CCGGCAGTCTCATTAGAGTG (SEQ ID NO: 6) (backward outer primer, 0.2 μM), CTCCTCCGTCTCCGGAGGATTCaaaaCGATGCAACGCGAAGAAC (SEQ ID NO: 7) (forward inner primer, 1.6 μM), TCGTCAGCTCGTGTCGTGAGATttttCCCAACCGAATGATGGCA (SEQ ID NO: 8) (backward inner primer, 1.6 μM), CGCACATGTCAAAACCAGG (SEQ ID NO: 9) (forward loop primer, 0.4 μM), and GCAACGAGCGCAACCC (SEQ ID NO: 10) (reverse loop primer, 0.4 μM). Equation 3 was used to compute percentage accessibility, where λ represents the template NA concentration in copies/μL as measured by dLAMP.

Ng culture preparation. Isolates were streaked from glycerol stocks stored at −80° C. onto BD BBL Chocolate II Agar plates and incubated overnight in a 37° C. incubator with 5% CO₂. Isolates were then passed onto fresh BD BBL Chocolate II Agar plates and grown for 4-7 h at 37° C. with 5% CO₂. In all experiments, cells from plates passed 1-3 times were used. Several colonies were scraped and resuspended in 37° C. GWM to generate a working suspension. Optical density at 600 nm (OD₆₀₀) was measured, and the working suspension was diluted to create a 2 mL working culture of OD₆₀₀ 0.05 in GWM in 15 mL polypropylene culture tubes. Cultures were incubated, with 500 rpm shaking, at 37° C.+5% CO₂ for 3-5 h prior to ABX exposure.

nuc-aAST time-course without enhancing step. Working cultures of Ng isolates were prepared as described in “Ng culture preparation.” Incubation at 37° C. was performed in 100 reaction volumes in PCR tube strips on a BioRad C1000 Thermal Cycler. Treated samples consisted of 77.5 μL MHB, 2.5 μL NaHCO₃ (200 mM), 5 μL DNase I (2 U/μL), 5 μL PEN (20 μg/mL), and 10 μL working Ng isolate culture. PEN was replaced with NF—H₂O in control samples. A 10 μL aliquot of each sample was extracted at 15, 30, 45, 60, 90, and 120 min and diluted 10× in QuickExtract DNA Extraction Solution (Lucigen, Middleton, Wis., USA), then heated for 6 min at 65° C. followed by 4 min at 98° C. on a BioRad C1000 Thermal Cycler. All sample handling following antibiotic exposure was performed using a multichannel pipette; qPCR and calculation of % accessibility were performed as described above.

Enhancer use. Working cultures of Ng isolates were prepared as described in “Ng culture preparation.” Initial exposure was performed by incubating 100 μL control and treated samples at 37° C. in PCR tube strips on a BioRad C1000 Thermal Cycler. Treated samples consisted of 75 MHB, 5 μL NaHCO₃ (100 mM), 5 μL DNase I (2 U/μL), 5 μL PEN or CRO (20 μg/mL), and 10 working Ng isolate culture. ABX were replaced with NF—H₂O in control samples. After 15 min of incubation, samples were vortexed and quick-spun, and aliquots of all samples were transferred to the enhancement step as described below. After the enhancement step, 5 or 10 μL of all samples were extracted by diluting 10× in QuickExtract DNA Extraction Solution (Lucigen) and heating for 6 min at 65° C. followed by 4 min at 98° C. on a BioRad C1000 Thermal Cycler. All sample handling following ABX exposure was performed using a multichannel pipette; qPCR and calculation of % accessibility was performed as described above.

Osmotic and autolytic enhancing steps were performed in 100 μL volumes. The osmotic enhancing step consisted of 89.75 μL NF—H₂O, 4.75 μL DNase I (2 U/uL), 0.5 μL CaCl₂ (100 mM, 0.2-μM filtered), and 5 μL initial exposure samples. The autolytic enhancing step consisted of 75 μL NF—H₂O, 4.75 μL NaHCO₃ (100 mM, 0.2-μM filtered), 10 μL Tris pH 8.5 (500 mM), 4.75 DNase I (2 U/uL), 0.5 μL CaCl₂ (100 mM), and 5 μL of the sample exposed to antibiotic.

All surfactant-enhancing steps were performed in 50 μL volumes with 25 of the 50 consisting of initial exposure samples. In the TNP enhancement step, the remaining 25 consisted of 1.25 μL DNase I (2 U/uL), 1.25 μL NaHCO₃ (100 mM), 20 μL MHB, and 2.5 TNP (100 mM). In the CHAPS enhancement step, the remaining 25 μL consisted of 1.25 DNase I (2 U/uL), 1.25 μL NaHCO₃ (100 mM), 20 μL MHB, and 2.5 μL CHAPS (200 mM). In the SDS and BAC enhancement steps, the remaining 25 μL consisted of 1.25 μL DNase I (2 U/uL), 1.25 μL NaHCO₃ (100 mM), 17.5 μL MHB, and either 5 μL SDS (1% w/v) or BAC (1% w/v) respectively.

Nuclease-accessibility AST validation. Working cultures were prepared, exposed to ABX, and enhancing steps performed as described for the CHAPS enhancement step in the “Enhancer use” section above. Extraction was performed as described above. Treated samples in the initial exposure step had a final concentration of 1.0 μg/mL PEN, CFM, or CRO. Samples were excluded if the percentage lysis (equation 2) due to CHAPS was >75%. If the percentage lysis was negative, the value was set to zero before averaging. Three to thirteen biological replicates were performed for each isolate-antibiotic combination. Biological replicates included separate antibiotic exposure, control exposure, and no-enhancer controls.

Preparation of clinical sample suspensions. Clinical urine samples were collected at the AIDS Healthcare Foundation (AHF) clinic under Caltech IRB #18-0865 from consented male patients symptomatic for N. gonorrhoeae. After initial collection by the patient, AHF research staff pipetted an 8-14 mL aliquot into a 15 mL conical tube. For each clinical sample, handling began within 30 min of the sample donation. A 1 mL aliquot of the clinical urine was centrifuged in a 2 mL screw-cap microcentrifuge tube (VWR) for 5 min at 1,000×g (Eppendorf 5418). The supernatant was then immediately removed, and the pellets resuspended in GWM to generate a working suspension. Next, a 320 μL aliquot of the working suspension was added to a mixture of 40 μL 10× DNase I Reaction Buffer (NEB), 20 DNase I (2000 U/mL) (NEB), and 20 Saponin (20% w/v; Cas #8047-15-2, TCI). The suspension was then vortexed, spun in a benchtop microcentrifuge at 2,000×g for 2-3 sec (Labnet Spectrafuge Mini Microcentrifuge), and placed on a heat block (GeneMate Mini Dry Bath) at 37° C. for 15 min. Next, the suspension was vortexed and centrifuged for 5 min at 1,000×g. The supernatant was removed, and the pellet resuspended in a mixture of equal volume and concentration of GWM, DNase I Reaction Buffer, and DNase I, as described above.

Clinical sample nuc-aAST. Suspensions of clinical urine samples were prepared as described above. All suspensions were generated, and the ABX-exposure step initiated within 90 min of sample donation. The initial ABX-exposure was performed by incubating 50 μL control and treated samples at 37° C. in PCR tube strips on a BioRad C1000 Thermal Cycler. Treated samples consisted of 48.8 μL of suspension and 1.25 μL aliquot of PEN or CRO (40 μg/mL). ABX were replaced with NF—H₂O in control samples. After 30 min of incubation, samples were vortexed and quickly spun on a benchtop microcentrifuge (Labnet) at 2,000×g for 2-3 sec, and 2.7 μL TNP (100 mM) was added to each sample for the enhancement step. Samples were then immediately vortexed, spun on a benchtop microcentrifuge (Labnet) at 2,000×g for 2-3 sec, and incubated at 37° C. for 3-5 min. After the enhancement step, 20 μL from each sample was extracted by diluting 5× in QuickExtract DNA Extraction Solution (Lucigen) and heating for 6 min at 65° C. followed by 4 min at 98° C. on a BioRad C1000 Thermal Cycler. All sample handling following ABX exposure was performed using a multichannel pipette; qPCR and calculation of percentage accessibility were performed as previously described, with the modification that the qPCR mix included 2 μL of template per 10 μL PCR reaction instead of 1 μL template per 10 μL reaction.

When processing the nucleic acid measurements, sample-inclusion criteria were as follows: samples must have had a 16S DNA concentration in the no-ABX control tube of less than a Cq of 29 (which translates to approximately 200 copies of 16S DNA/μL DNA extraction or 20 copies/μL in the PCR or LAMP reaction). ASTs from clinical samples with a negative percentage accessibility calculated to be less than −30% accessibility, or those with only one usable replicate out of three, were excluded from analysis.

MIC testing and creation of clinical isolates. While each of the clinical samples was being run with the nuc-aAST protocol, a 5-10 mL aliquot of the same clinical urine sample was packaged and transported on ice from AHF (Los Angeles, Calif.) to the laboratory at Caltech (Pasadena, Calif.). At the Caltech lab, a 50 μL aliquot was plated onto Neisseria-selective media (Modified Thayer Martin II (MTMII) Agar; Fisher Scientific) and incubated for 24-72 h at 37° C. and 5% CO₂. Four individual colonies were sub-cultured onto a fresh MTMII agar plate and incubated for 8-48 h at 37° C. and 5% CO₂. The agar plates were parafilm-sealed, packaged, and shipped overnight via FedEx at ambient temperature for isolation and gold-standard (agar-dilution) MIC testing at the Neisseria Reference Laboratory in Seattle, Wash. Agar-dilution MIC testing was performed as previously described.

MIC testing and creation of clinical isolates from clinical sample: While each of the clinical samples was being run with the nuc-aAST protocol, a 5-10 mL aliquot of the same clinical urine sample was packaged and transported on ice from AHF (Los Angeles, Calif.) to the laboratory at Caltech (Pasadena, Calif.). At the Caltech lab, a 50 μL aliquot was plated onto Neisseria-selective media (Modified Thayer Martin II (MTMII) Agar; Fisher Scientific) and incubated for 24-72 h at 37° C. and 5% CO2. Four individual colonies were sub-cultured onto a fresh MTMII agar plate and incubated for 8-48 h at 37° C. and 5% CO2. The agar plates were parafilm-sealed, packaged, and shipped overnight via FedEx at ambient temperature for isolation and gold-standard (agar-dilution) MIC testing at the Neisseria Reference Laboratory in Seattle, Wash. Agar-dilution MIC testing was performed as previously described.

Ng suspensions from clinical isolate to compare to clinical sample: A 1 mL aliquot of the clinical isolate in culture media, prepared was made according to the methods described for “NG Stock and Resuspension”. This aliquot was centrifuged in a 2 mL screw-cap microcentrifuge tube (VWR) for 5 min at 1,000×g (Eppendorf 5418). The supernatant was then immediately removed, and the pellets resuspended in GWM to generate a working suspension. Next, a 320 μL aliquot of the working suspension was added to a mixture of 40 μL 10× DNase I Reaction Buffer (NEB), 20 μL, DNase I (2000 U/mL) (NEB), and 20 μL Saponin (20% w/v; Cas #8047-15-2, TCI). The suspension was then vortexed, spun in a benchtop microcentrifuge at 2,000×g for 2-3 sec (Labnet Spectrafuge Mini Microcentrifuge), and placed on a heat block (GeneMate Mini Dry Bath) at 37° C. for 15 min. Next, the suspension was vortexed and centrifuged for 5 min at 1,000×g. The supernatant was removed, and the pellet resuspended in a mixture of equal volume and concentration of GWM, DNase I Reaction Buffer, and DNase I, as described above.

Clinical sample nuc-aAST repeated with clinical isolates. After the clinical isolate was prepared and the gold-standard agar-dilution MIC test was run, the isolate was shipped back from the Neisseria Reference Laboratory (Seattle, Wash.) to the Caltech laboratory (Pasadena, Calif.). The isolate was then grown according to the methods for “Ng culture preparation” previously described. The experimental steps for “Clinical sample preparation” and “Clinical sample nuc-aAST” were repeated using the cell suspension of the isolate instead of the urine sample. The data from these clinical isolates were then compared to the results of the nuc-aAST performed directly on the original clinical urine samples.

Timed sum-of-steps. Working cultures of Ng isolates used in FIG. 58 were prepared as described in “Ng culture preparation” and 1.5 mL of the cultures were pelleted at 2500 g for 2.5 min and resuspended in 150 μL normal human urine (Lee Biosciences) pre-warmed to 37° C. Initial exposure was performed by incubating 100 μL control and treated samples at 37° C. in PCR tube strips on a BioRad C1000 Thermal Cycler. Treated samples consisted of 65 μL MHB, 5 NaHCO₃ (100 mM), 5 μL DNase I (2 U/μL), 5 μL PEN (20 μg/mL), and 20 μL Ng isolate suspension in urine. NF—H₂O was used in place of PEN in control samples. A CHAPS enhancing step was performed as described above. After the enhancement step, a 20 μL aliquot from each sample was extracted by diluting 5× in QuickExtract DNA Extraction Solution (Lucigen) and heated for 1 min at 65° C. followed by 1 min at 98° C. on a BioRad C1000 Thermal Cycler. All sample handling following ABX exposure was performed using a multichannel pipette. Amplification was then performed using qPCR, ddPCR, or dLAMP. Extractions were diluted 2.5× in NF—H₂O before use in dLAMP.

Osmolarity measurements. Osmolarity measurements were performed on a Model 3320 Osmometer (Advanced Instruments Inc., Norwood, Mass., USA). The instrument was calibrated with reference standards (Advanced Instruments) prior to experiments. Samples identical to the antibiotic-exposure condition (i.e. media, nuclease, etc.) and samples identical to the osmotic enhancing condition were prepared and measured. The volume that would normally be comprised of Ng culture was replaced with media.

Statistical analysis. P-values for FIG. 56 were calculated using GraphPad Prism 8.0 software from an unpaired, two-tailed t-test comparing the averages of the three replicates of each susceptible sample to each resistant sample. A significance value of 0.02 was used for statistical significance. ROC plots used for setting susceptibility thresholds in FIG. 58 were created using GraphPad Prism 8.0 software. Sensitivity was defined as the proportion of gold-standard susceptible samples correctly identified as susceptible by the nuc-aAST. Specificity was defined as the proportion of gold-standard resistant samples correctly identified as resistant by the nuc-aAST. Statistical analyses for FIG. 60, (dLAMP measurements) were performed as published previously [2, 16]. As in the previous publication [9], the control and treated concentrations are compared as a ratio for statistical analysis.

$\begin{matrix} {{{Concentration}\mspace{14mu}{Ratio}} = \frac{\lambda_{control}}{\lambda_{treated}}} & (7) \end{matrix}$

This concentration ratio is transformed into a percentage change for visualization purposes, but the ratio is assessed for statistical significance. Poisson statistics were used to calculate the confidence interval of the NA concentration for each measurement. The error in the concentration ratio, a term used in the calculation of percentage accessibility, is calculated with standard-error propagation methods:

$\begin{matrix} {\sigma_{ratio} = \sqrt{\left( \frac{\sigma_{\lambda_{2}}}{\lambda_{1}} \right)^{2} + \left( \frac{\lambda_{2} \cdot \sigma_{\lambda_{1}}}{\lambda_{1}^{2}} \right)^{2}}} & (8) \end{matrix}$

A one-tailed Z-test, assuming a normal distribution, is used to calculate p-values for digital NA concentrations. A threshold value for significance is set as a ratio of 1.22, corresponding to a percentage accessibility of 18%.

$\begin{matrix} {Z = \frac{{\ln\;\left( l_{co{ntrol}} \right)} - {\ln\;\left( {1.22l_{treated}} \right)}}{\sqrt{{\sigma^{2}in_{(\lambda_{control})}} + {\sigma^{2}in_{(\lambda_{treated})}}}}} & (9) \end{matrix}$

A significance value of 0.05 was used for statistical significance. The p-values to determine significance in dLAMP experiments were computed using Microsoft Excel's standard normal cumulative distribution function and Z-value.

DNase I inactivation experiment. Incubations were performed in PCR tubes on a Bio-Rad C1000 Thermal Cycler. Samples including DNase I (Samples B-F in FIG. 61) consisted of 90 μL MHB, 5 μL NaHCO₃ (100 mM), and 5 μL NEB DNase I (2000 U/mL). Nuclease-free water was used in place of DNase I in the control sample (Sample A in FIG. 61). After vortexing to mix, 5 μL of each suspension was added to 45 μL QuickExtract DNA Extraction Solution (Lucigen). Samples A and B were heated at 65° C. for 6 min and 98° C. for 4 min. Sample C was heated at 65° C. for 4 min and 98° C. for 3 min. Sample D was heated at 65° C. for 3 min and 98° C. for 2 min. Sample E was heated at 65° C. for 2 min and 98° C. for 1 min. Sample F was heated at 65° C. for 1 min and 98° C. for 1 min. After each heating step was completed, the samples were placed on an ice block. DNase I activity was tested by adding 15 μL of a spike-in control of Ng DNA (1/20 dilution of stock Cq 19, expected concentration in PCR in this experiment to be a Cq of 23.3) to 15 μL of the Samples A-F in PCR tubes, samples were heated to 37° C. for 5 min, and then the samples were quantified in qPCR, as described in the methods section above.

DNA digestion of the lambda spike-in. Seven or eight 1.25 mL aliquots of clinical urine samples were centrifuged in 2 mL microcentrifuge tubes (VWR) for 5 min at 1,000×g (Eppendorf 5418R). Pellets were re-suspended in 250 μL GWM containing a lambda DNA spike-in (see below for preparation). All working suspensions were then pooled together. To generate the (+) DNase I controls, 320 μL aliquots of the working suspensions were added to 40 μL 10× DNase I Reaction Buffer (NEB), 20 μL DNase I (2,000 U/mL) (NEB), and 20 μL nuclease-free water. Nuclease-free water was used in place of DNase I in the (−) DNase I control sample. Suspensions were then vortexed, quickly spun on a benchtop microcentrifuge (Labnet) at 2,000×g for 2-3 sec, and incubated at 37° C. for 15 min on a heat block (ThermoScientific Digital Shaking Drybath). Suspensions were then removed from the heat block, vortexed, and spun on a benchtop microcentrifuge (Labnet) at 2,000×g for 2-3 sec. After mixing, 20 μL of each suspension was added to 80 μL QuickExtract DNA Extraction Solution (Lucigen). Samples were heated at 65° C. for 6 min and 98° C. for 4 min on a BioRad C1000 Thermal Cycler. DNase I activity was tested by subtracting the concentration of lambda DNA in the (−) DNase I control from the concentration in the (+) DNase I control tube. Quantification of DNA was done with qPCR (Roche, LightCycler 96) as described in the methods above. As in the clinical nuc-aAST, 2 μL of template per 10 qPCR reaction was used.

The lambda DNA spike-in was prepared as follows. 12.5 μL lambda phage DNA (7.1×10⁵ copies/μL, as quantified with ddPCR) (QX200, Bio-Rad) in 0.5×TE Buffer was mixed with 1238 μL Graver-Wade Medium (GWM).

Example 0: Modification of Nucleic Acid Accessibility in a Cell Following Administration of an Antibiotic

The general methods and material indicated above were used in ASTs in which detection of accessible and inaccessible nucleic acid is set up based on the modification in nucleic acid accessibility of nucleic acid associated with antibiotic administration schematically illustrated in FIGS. 1 and 2.

In particular the schematic illustration of FIG. 1 shows difference in accessibility and a corresponding exemplary outcome of an exemplary antibiotic susceptibility test for a susceptible microorganism of the instant disclosure. In particular, FIG. 1 Panel A shows a schematic representation of a control sample comprising the microorganism not treated with antibiotic and showing inaccessible DNA in an intact cell. FIG. 1 Panel B shows a schematic representation of an antibiotic-treated sample showing a disrupted or lysed susceptible microorganism cell with DNA accessible to nuclease. FIG. 1 Panel C shows a diagram illustrating the CT ratio of the control sample of Panel A and the antibiotic-treated sample of FIG. 1 Panel B. A threshold control-treated (CT) ratio (the dashed line with a prediction of the cell being antibiotic susceptible (S).

The illustration of FIG. 2 shows instead a schematic representation of difference in nucleic acid accessibility and a corresponding exemplary outcome of an exemplary antibiotic susceptibility test for a resistant microorganism of the instant disclosure. In particular, FIG. 2, Panel A shows a schematic representation of a control sample comprising the microorganism not treated with antibiotic and showing inaccessible DNA in an intact cell. FIG. 2, Panel B shows a schematic representation of an antibiotic-treated sample showing intact resistant microorganism cell. FIG. 2, Panel C shows a diagram illustrating the CT ratio of the control sample of Panel A and the antibiotic-treated sample of Panel B. A threshold control-treated (CT) ratio (the dashed line with a prediction of the cell being antibiotic resistant (R).

Example 1: AST Performed by Detecting Inaccessible DNA with 1 Incubation Step (Media with Enhancing Treatment; Followed by Cell Lysis and NA Extraction) with DNA Quantification Performed by qPCR

An antibiotic susceptibility test can be performed with methods herein described comprising 15-minute co-incubation of the sample with antibiotics and an enhancing treatment followed by cell lysis, and detection of differences in amounts of detected DNA between the antibiotic-susceptible and a corresponding control but not in antibiotic-resistant NG isolates with respect to a corresponding control.

In particular this example shows a detection of a difference in amounts of DNA in a treated incubation for an antibiotic-susceptible (ng_3, ceftriaxone susceptible) clinical isolate with respect to a corresponding control, but not in antibiotic-resistant (ng_30, ceftriaxone resistant/reduced-susceptible) clinical isolate.

In particular, the detection was performed in outcome of to the following procedure.

1. Provide Incubation Media Containing Enhancer, with and without Antibiotic for Each Isolate

Several milliliters of media were prewarmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods) for ≥30 minutes prior to use.

For each isolate, two incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the indication in table x below, titled “Antibiotic Exposure per 500 μL”. Add Triton X (enhancer), DNaseI (Cat: M0303L) (degrader), NaHCO₃ (conditioner).

Antibiotic was added when bacteria cells were incubating, so that the time from thawing the antibiotic stock was minimized before exposure (see step 3). In particular antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks: Diluted 1 mg/mL ceftriaxone stock, prepared according to “Preparing Antibiotic Stocks” description above, see ceftriaxone. The stocks used had short-term storage of the 1 mg/mL stocks in −20° C. storage instead of −80° C. storage, but were only thawed once. To Prepare 50× stocks of the antibiotic follow the following dilutions in nuclease-free (NF) water:

-   -   50× Stock (1×4 μg/mL cro→200 μg/mL=0.2 mg/mL cro)         -   400 μL of 0.2 mg/mL=80 μL (1 mg/mL stock)+320 μL NF water     -   50× Stock (1×0.25 μg/mL cro→12.5 μg/mL=0.0125 mg/mL cro)         -   400 μL of 0.0125 mg/mL=62.5 mg/mL dilution)+337.5 μL NF             water

The resulting mixture was thus provided

Antibiotic Exposure per 500 μL. (2 mL purple cap tubes setup) Component Volume Ng Suspension (OD = 0.5) 50 μL (OD = 0.05) MHB (Prewarmed) (media) 360 μL 10X detergent (10% TritonX) (enhancer) 50 μL (1X, 1%) NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) Ceftriaxone (50X), see ABX dilutions 10 μL (1X) above (treated) or replace with nuclease- free (NF) Water in controls (untreated) NaHCO₃ (100 mM) (conditioner) 5 μL (1 mM)

This step can be performed before or after steps 2-4 of this example as will be understood by a skilled person upon reading of the present disclosure.

2. Provide a Sample Containing Bacteria

The bacteria were prepared according to the “NG Stock and Resuspension” description above with the following changes: MHB+5 mM NaHCO₃ was shaken at 800 rpm for 55 min in the incubator, p0 cells were used, 50 minutes of resuspension in MHB at 37° C., 5% CO2, 1000 rpm, and the cell clumps were not manually broken apart with a pipette.

While cell pellet was suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared.

Right before measuring OD, of step 3 of this example, the antibiotic exposure tubes prepared in step 1 were set on the shaking heat block (set to 38° C. and 1000 rpm shaking)

3. Add the Antibiotic to the Exposure Tube

In variation of this set of experiments where the antibiotic was not added during step 1, the antibiotic exposure tubes was brought from the shaking heat block (set to 38° C. and 1000 rpm shaking) to the BSC. 10 μL of the appropriate antibiotic dilution was added to the exposure tubes. For the exposure tubes which was set to go “untreated” 10 μL of NF water was added.

4. Optionally, Pre-Process the Sample Containing the Bacteria to Modify the Bacterial Concentration in the Sample

In a variation of the set of experiments herein described the OD600 of each of the suspensions prepared in step 2 can be adjusted to OD600=0.5. Measure the OD600 (dilute 1/10 in pre-warmed media) and dilute accordingly in pre-warmed media.

5. Split the Sample into Two or More Parts

50 μL of the Ng suspension (such as the OD 0.5 suspensions prepared in step 4) was withdrawn.

6. Combine the Sample with Incubation Media

50 μL of the Ng suspension was added into “treated” media, and 50 μL of the Ng suspension was added into untreated media. This addition can be performed sequentially or in parallel (using multichannel pipet) as will be understood by a skilled person upon reading of the present disclosure

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time.

The antibiotic exposure tubes prepared in step 1 were returned on the shaking heat block (set to 38° C. and 1000 rpm shaking).

The antibiotic incubation/exposure was performed on a heat-shake block fit to 2 mL purple cap tubes (VWR Cat #89004-298). The particular shake-block was measured with a thermocouple to ensure the temperature inside the tubes with 500 μL of media is 37° C. (settings 38° C. and 1000 rpm shaking). qPCR was used for NA amplification

The liquid temperature of this particular heat/shake block was measured with a thermocouple to ensure the temperature inside the tubes with 500 μL of media is 37° C. (settings: 38° C. and 1000 rpm shaking).

8. Extraction (at 15 Minutes) of Treated and Untreated Samples to Purify DNA

Extraction was performed following “DEB Extraction” description in the general methods using Eppendorf tubes. Vortex samples briefly (2 seconds) and centrifuge before transferring 10 μL of the sample to 90 μL of the extraction buffer.

9. Optionally, Continue Incubation of the Treated and Untreated Samples for a Control of the Antibiotic Efficacy Over >15 Minute Exposures

After desired incubation time >60 minutes, 5 μL droplets were plated on a pre-warmed BBL Chocolate Agar plate as a positive control of the antibiotic.

10. Measurement of DNA by qPCR

The “qPCR” description in the general methods was followed. All DEB-extracted DNA were quantified using a primer mix=(10 μM forward and reverse) targeting N. gonorrhoeae porA gene. Samples were run in qPCR triplicates (10 μL reactions) at an annealing temperature 62° C. The primers target the porA gene.

11. Data Analysis is Performed on the Raw Cq Measurements Obtained from the qPCR Instrument

The related results are illustrated in FIG. 3 which illustrates the results for one ceftriaxone-susceptible Ng isolates (ng_3) (se FIG. 3, (a) and (b) bars) and one ceftriaxone-resistant Ng isolate (ng_30) (see FIG. 3, (c) and (d) bars). Cq's detected plotted with error bars of 95% confidence intervals based on the qPCR triplicate measurements.

As can be seen from the illustration of FIG. 3, upon incubation with 1% TritonX-100, NEB DNaseI, and ceftriaxone ABX in cell culture media (MH), the susceptible isolates showed a decrease in DNA concentration. This is likely due to an increased accessibility of the DNA due to lysed or disrupted cells. The DNaseI added to the incubation digests the accessible DNA. The increase in Cq corresponds to decreased quantity of detectable inaccessible nucleic acids. These data also show that increasing antibiotic concentration can enhance the effect of a beta-lactam treatment after a short (15-minute) incubation time. This effect is not seen in the resistant isolate, as the Cqs are not distinguishable from the control (no ABX). Note that 3.5 Cq difference between FIG. 3 bars (a) and (b) on the exponential scale represents an 11-fold difference in relative concentrations.

The results exemplified in FIG. 3, support the conclusion that a 15-minute co-incubation with antibiotics and a non-ionic surfactantenhancer of 1% Triton X-100 show differences in DNA accessibility (as seen by the difference in Cq) between the antibiotic-susceptible but not antibiotic-resistant NG isolates.

The results exemplified in FIG. 3, also supports the conclusion that the difference in Cqs (detectable difference) is sufficient to differentiate between the antibiotic-susceptible and antibiotic-resistant isolates of NG.

The results exemplified in FIG. 3, further support the conclusion that same protocol can be run on isolates on different days, and the results can be compared between days.

Example 2: AST Performed by Detecting Inaccessible DNA with 1 Incubation Step (Media with Enhancer; Followed by Cell Lysis and NA Extraction) with DNA Quantified with qPCR and Treatment with Antibiotic Concentration Above Breakpoint to Shorten the Exposure Time

Experiments were performed to show that antibiotic concentration above the resistance breakpoints can be used with short antibiotic exposures in AST performed with methods herein described. In particular in this example AST was performed by 30-minute co-incubation with antibiotics and an enhancer followed by cell lysis, nucleic acid extraction and detection in detectable differences in DNA between the antibiotic-susceptible but not antibiotic-resistant NG isolates.

The procedure used in this example is identical to the one of Example 1, except for the fact that the isolates here are one ceftriaxone-susceptible Ng isolates (ng_17) and one ceftriaxone-resistant Ng isolate (ng_30), and for the fact that the antibiotic exposure is 30 minutes instead of 15 minutes.

The related data are shown in FIG. 4 which illustrates the results for the ne ceftriaxone-susceptible Ng isolates (ng_17) (see FIG. 4, (a) (b) and (c) bars) and the one ceftriaxone-resistant Ng isolate (ng_30) (see FIG. 4, (d) (e) and (f) bars). Primers target the porA gene. Cq's are plotted with error bars of 95% confidence intervals based on the qPCR triplicate measurements.

As can be seen from the illustration of FIG. 4, upon incubation with 1% TritonX-100, NEB DNaseI, and ceftriaxone ABX in cell culture media (MH), the susceptible isolates showed a decrease in DNA concentration.

The exemplary results illustrated in FIG. 4, show that treating with a concentration of antibiotic even one that is higher than the breakpoint of ceftriaxone can enhance the effect seen at an exposure times without observing an increase in DNA accessibility for the resistant isolate. This figure shows that the method herein described allow a measurable effect from the susceptible-treated condition with a shorter treatment.

The exemplary results illustrated in FIG. 4, show that 30-minute co-incubation with antibiotics and an enhancer of 1% Triton X-100 show differences in DNA accessibility (as seen by the difference in Cq) between the antibiotic-susceptible but not antibiotic-resistant NG isolates. The increase in Cq corresponds to decreased quantity of detectable inaccessible nucleic acids. The differences are larger in the ceftriaxone-susceptible isolate when the antibiotic concentration was increased without having a complimentary effect in the antibiotic-resistant isolate. Accordingly, the difference in Cqs as measured with qPCR is sufficient to differentiate between the antibiotic-susceptible and antibiotic-resistant isolates of NG.

The exemplary results illustrated in FIG. 4, support the conclusion that with a given antibiotic-exposure time, increasing the antibiotic concentrations can yield a larger difference in nucleic acid accessibility (shown with the difference of Cqs between the treated and untreated samples) as will be understood by a skilled person.

Example 3: AST Performed by Detecting Inaccessible DNA with 2 Incubation Steps (Media; Dilute into Enhancing Buffer; Followed by Cell Lysis and NA Extraction) with DNA Quantified by qPCR

Antibiotic susceptibility test was performed with methods herein described comprising 2 sequential incubation steps, and in particular

a primary incubation in which a sample is contacted with media, antibiotic (or water in untreated sample), and a nuclease (such as DNaseI), and optionally an enhancer (such as 1% Triton X-100); and

a secondary incubation in which a cell suspension from primary incubation is contacted with enhancers such as 50 mM TRIS buffer at pH 8.5 and/or 1% Triton X-100, possibly using dilution of the cell suspension from the antibiotic exposure by 90-95% (such as 50 μL into 450 μL, 25 μL into 475 μL) removing most antibiotic, culture media, and other components from the primary incubation for the enhancer condition.

In particular this example shows a detection of a difference in amounts of DNA in a treated incubation for an antibiotic-susceptible (ng_17, ng_3, penicillin-susceptible) clinical isolate with respect to a corresponding control, but not for a antibiotic-resistant (ng_44, penicillin resistant) clinical isolate.

In particular, the detection was performed in outcome of the following procedure.

1. Provide Incubation Media Containing Enhancer, with and without Antibiotic for Each Isolate

Several milliliters of media were prewarmed in a 37° C., 5% CO₂ incubator (media is HFB, see “HFB media preparation” in general methods) for ≥15 minutes prior to use. Two milliliter aliquots were used for each isolate and addition media is needed for OD600 measurements and dilutions (see step 4 of this example)

For each isolate, incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the chart below, titled Primary incubation 1 or 2. For each of the desired incubation conditions, an untreated and treated tube were used (all comparisons are made from identical handling conditions; e.g. the same primary and secondary incubation conditions and dilutions). Add Triton X (enhancer), DNaseI (Cat: M0303L) (degrader) as instructed by the tables below. Antibiotic was added when bacteria cells were incubating, so that the time from thawing the antibiotic stock was minimized before exposure (see step 3 of this example).

Primary incubation (option 1) Antibiotic Exposure per 500 μL. (2 mL purple cap tubes setup) Component Volume Ng Suspension (OD600 = 0.5 in HFB, media) 465 μL NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) Penicillin (50X), see ABX dilutions in 10 μL (1X) step 3 (treated) or replace with nuclease-free (NF) Water in controls (untreated)

Primary incubation (option 2) Antibiotic Exposure per 500 μL (±) Triton X-100. Volume from Triton X-100 replaced with NF water in this control (2 mL purple cap tubes setup) Component Volume Ng Suspension (OD600 = 0.5 in HFB, media) 50 μL 10X detergent (10% TritonX) (enhancer) 50 μL (1X, 1%) or replace with nuclease-free (NF) water in controls (control, in place of enhancer) NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) Ceftriaxone (50X), see ABX dilutions above 10 μL (1X) (treated) or replace with nuclease-free (NF) Water in controls (untreated)

For each isolate, incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the chart below, titled Secondary incubation 1, 2, or 3. These conditions were set up for a sequential incubation containing enhancers.

Secondary incubation (option 1) Enhancer per 500 μL TRIS pH 8.5. (2 mL purple cap tubes setup) Component Volume Ng Suspension from antibiotic exposure 25 μL 10X TRIS pH 8.5 (500 mM) (enhancer) 50 μL (1X, 50 mM) NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) CaCl₂ (50 mM) 5 μL (1X, 0.5 mM) NaHCO₃ (100 mM) 5 μL (1 mM) NF water 390 μL

Secondary incubation (option 2) Enhancer per 500 μL TRIS pH 8.5. (2 mL purple cap tubes setup) Component Volume Ng Suspension from antibiotic exposure 50 μL 10X TRIS pH 8.5 (500 mM) (enhancer) 50 μL (1X, 50 mM) NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) CaCl₂ (50 mM) 5 μL (1X, 0.5 mM) NaHCO₃ (100 mM) 5 μL (1 mM) NF water 365 μL

Secondary incubation (option 3) Enhancer per 500 μL TRIS pH 8.5 + 1% Triton X-100. (2 mL purple cap tubes setup) Component Volume Ng Suspension from antibiotic exposure 50 μL 10X TRIS pH 8.5 (500 mM) (enhancer) 50 μL (1X, 50 mM) 10X detergent (10% TritonX) (enhancer) 50 μL (1X, 1%) or replace with nuclease-free (NF) water in controls (control, in place of enhancer) NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U/mL) CaCl₂ (50 mM) 5 μL (1X, 0.5 mM) NaHCO₃ (100 mM) 5 μL (1 mM) NF water 315 μL

This step 1 can be performed before or after steps 2-4 of this example as will be understood by a skilled person upon reading of the present disclosure.

2. Providing a Sample Containing Bacteria

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section with the following changes: media used was HFB shaken at 1000 rpm for 54-65 min in the incubator, p0 cells were used, 30-80 minutes of resuspension in HFB at 37° C., 5% CO2, 1000 rpm, and the cell clumps were not manually broken apart with a pipette.

While cell pellet was suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared.

Right before measuring OD, step 3, the primary incubation and secondary incubation tubes prepared in step 1 were set on the shaking heat block (set to 38° C. and 1000 rpm shaking) to warm the contents to 37° C.

3. Add the Antibiotic to the Exposure Tube

Antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks: Diluted 1 mg/mL ceftriaxone stock, according to “Preparing Antibiotic Stocks” description in the general methods, see ceftriaxone. The stocks used in this example had short-term storage of the 0.05 mg/mL stocks in −20° C. storage instead of −80° C. storage. No further dilutions needed to be made, since this concentration is at 50× working concentration.

The antibiotic incubation/exposure was performed on a heat-shake block fit to 2 mL purple cap tubes (VWR Cat #89004-298). The particular shake-block was measured with a thermocouple to ensure the temperature inside the tubes with 500 μL of media is 37° C. (settings 38° C. and 1000 rpm shaking).

The primary incubation (antibiotic exposure) tubes were brought from the shaking heat block (set to 38° C. and 1000 rpm shaking) to the BSC. Add 10 μL of the appropriate antibiotic dilution to the exposure tubes. For the exposure tubes which will go “untreated” add 10 μL of NF water.

The secondary incubation (enhancer) tubes were set on the shaking heat block (set to 38° C. and 1000 rpm shaking).

4. Optionally, Pre-Process the Sample Containing the Bacteria to Modify the Bacterial Concentration in the Sample

In a variation of this set of experiment the OD600 of each of the suspensions prepared in step 2 can be adjusted to OD600=0.5. Measure the OD600 (dilute 1/10 in pre-warmed media) and dilute accordingly in pre-warmed media.

5. Split the Sample into Two or More Parts

50 μL or 465 μL of the Ng suspension (such as the OD 0.5 suspensions prepared in step 4 of this example) were withdrawn. The incubation charts in step 1 was followed to set up the volumes to be added to the corresponding tubes.

6. Combine the Sample with Incubation Media

50 μL or 465 μL of the Ng suspension were added into “treated” media, add 50 μL or 465 μL of the Ng suspension into untreated media. Note this addition can be performed sequentially or in parallel (using multichannel pipet).

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time.

The antibiotic exposure tubes prepared in step 1 were returned on the shaking heat block (set to 38° C. and 1000 rpm shaking). T the liquid temperature of this particular heat/shake block was measured with a thermocouple to ensure the temperature inside the tubes with 500 μL of media is 37° C. (settings: 38° C. and 1000 rpm shaking).

8. Dilution of Treated and Untreated Samples (at 30 Minutes) into a Secondary Incubation for a Short Amount of Time

The samples were mixed before transferring 25 μL or 50 μL of the sample to the prepared secondary incubation (enhancer) tubes. The charts in step 1 were followed to add the appropriate volume of cells to each tube. Mix the tubes and proceed to step 9 immediately

9. Extraction (at 30 Minutes) of Treated and Untreated Samples to Purify DNA

Extraction was performed following the “DEB Extraction” description in the general methods using Eppendorf tubes. Mix the samples that have just been diluted into the secondary incubation tubes in step 8 before transferring 10 μL of the contents to 90 μL of the extraction buffer.

The secondary incubation (enhancer) tubes were returbed to the shaking heat block to continue incubation at 37° C.

10. Extraction (at 50 Minutes) of Treated and Untreated Samples to Purify DNA

Extraction was performed following the “DEB Extraction” description in the general methods using Eppendorf tubes. The samples from the secondary incubation tubes in step 9 of this example were mixed before transferring 10 μL of the contents to 90 μL of the extraction buffer

11. Optionally, Continue Incubation of the Treated and Untreated Samples from the Primary Incubation for a Control of the Antibiotic Efficacy Over >30 Minute Exposures

In a variation of the procedure exemplified herein after desired incubation time >60 minutes, 5 μL droplets can be plated on a pre-warmed BBL Chocolate Agar plate as a positive control of the antibiotic.

12. Measurement of DNA by qPCR

Detection of DNA was performed following the “qPCR” description in the general methods. Quantify all DEB-extracted DNA using the Primer mix=(10 μM forward and reverse) targeting N. gonorrhoeae porA gene. Samples were run in qPCR triplicates (10 μL reactions) at an annealing temperature 62° C. The primers did target the porA gene.

13. Data Analysis is Performed on the Raw Cq Measurements Obtained from the qPCR Instrument

The results illustrated in FIG. 5 show differences in responses which can be seen by comparing the time 30 and time 50 measurements from the second incubation step in TRIS buffer pH 8.5. All samples are expected to have an increased rate of lysis (represented here as the reciprocal of the fold change, to visualize positive values). Cq's are plotted with error bars of 95% confidence intervals based on the qPCR triplicate measurements. The increase in Cq corresponds to decreased quantity of detectable inaccessible nucleic acids.

This observation is confirmed by data obtained by the exemplified method with the inclusion of an additional enhancer (1% Triton X-100 in this example) during both the primary incubation and secondary incubation are shown in FIG. 6. FIG. 6 shows how enhancers might be combined to get a larger change in detectable nucleic acids, since an increase in Cq in the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids.

The illustration of FIG. 5 and FIG. 6 clearly illustrate the increase in partial lysis, measured by DNA accessibility, after 50 minutes of incubation (30 min ABX exposure in cell culture media followed by 20 minutes of an enhancing treatment in TRIS buffer), nucleic acid extraction follows this exposure, and quantification is done with qPCR. Quantification could be done with several different methods. One could draw a threshold line for future comparisons of susceptible and resistant responses.

These data are obtained from steps 1-13 of this example with primary incubation #2 and secondary incubations #2 and #3. The interpretation of susceptibility could be determined from the magnitude of the 1/FC plots by the treated samples only. (In the above example the fold change of the susceptible-treated samples is >5 and the fold change of the resistant, and each of the controls is approximately 1.5)

The results shown in FIG. 5 and FIG. 6 supports the conclusion that the two-step incubations with enhancers in the secondary incubation only increase DNA accessibility in the susceptible-treated isolate after short beta-lactam exposures. Accordingly, the results of these examples support the conclusion that two-step incubation with enhancers in both the primary and secondary incubations increase DNA accessibility in the susceptible-treated samples.

Example 4: AST Performed by Detecting Inaccessible DNA with 1 Step Incubation with an Enhancer and β-Lactams Followed by Cell Lysis and DNA Extraction with DNA Quantified with Digital PCR

Antibiotic susceptibility test was performed with methods herein described comprising one-step incubation with an enhancer with three different beta-lactam antibiotics.

In particular, this example shows a detection of a difference in amounts of DNA in a treated incubation for an antibiotic-susceptible clinical isolate with respect to a corresponding control, but not for an antibiotic-resistant clinical isolate.

In particular, the isolates ng_3, ng_15, ng_17, ng_18, and ng_30 were used (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods). Penicillin was tested on isolates ng_15 (PEN-S) and ng_18 (PEN-R) and used the media HFB. Ceftriaxone was tested on isolates ng_17 (CRO-S) and ng_30 (CRO-R) and used the media MHB. Cefixime was tested on isolates ng_3 (CFM-S) and ng_30 (CFM-R) and used the media MHB.

The DNA detection was performed in outcome of the following procedure.

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters of media were pre-warmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods; or HFB) for ≥30 minutes prior to use. The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section with the following changes: In this example, the penicillin exposures used HFB and the ceftriaxone and cefixime exposures used MHB.

For each isolate, two incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the chart below, titled “Antibiotic Exposure per 500 μL”. Add Triton X (enhancer), DNaseI (degrader), NaHCO₃ (conditioner, if using MHB as the media). Antibiotic was added when bacteria cells were incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 4 of this example).

Table of components for media preparation and antibiotic exposure per 500 μL (2 mL purple cap tubes setup) Component Volume Media (Prewarmed HFB or MHB) (media) 360 μL Triton X-100 detergent (10X, 10%) (enhancer) 50 μL (1X, 1%)    NEB DNaseI (2000 U/mL) (degrader) 25 μL (100 U · mL) Antibiotic (50X), see ABX dilutions in step 4 10 μL (1X)      (treated) (OR replace with NF Water in controls, (untreated) NaHCO₃ (100 mM) 5 μL (1 mM)    

Table of components for media preparation and antibiotic exposure per 500 μL (2 mL purple cap tubes setup) Component Volume Ng Suspension (OD = 0.5) 50 μL (OD = 0.05)

2. Providing a Sample Containing Bacteria

Cell pellets were resuspended and in particular cell pellet were scraped with inoculating loop, twirled in 2 mL media and incubated on shaker (1000 rpm) in 37° C., 5% CO₂ incubator (≥30 minutes).

While cell pellet was suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared.

Right before measuring OD, these tubes were set on the shaking heat block (set to 38° C. and 1000 rpm shaking)

3. Optionally, Pre-Process the Sample Containing Bacteria

In a variation of the procedure exemplified in this example, the OD600 (dilute 1/10 in media) was measured, OD's below were corrected for the 1/10 dilution for the measurement. ng_30 OD=1.1 (before dilution). Adjust OD of suspension to OD=0.5 in prewarmed MHB+5 mM NaHCO₃ media

4. Add Antibiotic to the Exposure Tubes

Antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks as follows: Diluted 1 mg/mL ceftriaxone stock, was prepared according to “Preparing Antibiotic Stocks” description in the general methods, see ceftriaxone. To Prepare 50× stocks of the antibiotic the following dilutions were performed: Dilutions from 1 mg/mL Penicillin stock (aliquot from ES−20° C. box 2 as prepared on 20170822_1) to (50× antibiotic) as indicated below for each antibiotic.

-   -   Penicillin: 4 μg/mL (1×)→200 μg/mL (50×)=0.2 mg/mL         -   200 μL of 50× stock=40 μL 1 mg/mL stock+160 μL NF water in             nuclease-free (NF) water     -   Ceftriaxone: 4 μg/mL (1×)→200 μg/mL (50×)=0.2 mg/mL         -   200 μL of 50× stock=40 μL 1 mg/mL stock+160 μL NF water in             nuclease-free (NF) water     -   Cefixime: 1×3 μg/mL 4 μL 50× Stock (1×3 μg/mL cro→150 μg/mL=0.15         mg/mL cro)         -   500 μL of 0.150 mg/mL=75 μL (1 mg/mL stock)+425 μL NF water

Antibiotic was added to the “treated” sample. Nuclease free water was added to the untreated sample.

5. Split the Sample into Two or More Parts

50 uL of the Ng suspension were withdrawn into a pipette

6. Combine Sample with Incubation Media

50 uL of the Ng suspension were added into “treated” media, and 50 uL of the Ng suspension were added into the untreated media. This addition can be performed sequentially or in parallel (using multichannel pipet) as will be understood by a skilled person

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

The treated and untreated samples were incubated at 37 C for 15 minutes

8. Extraction (at 15 Minutes) of Treated and Untreated Samples to Purify RNA and DNA

Extraction was performed following the procedure outlined herein “DEB Extraction” in the general methods section, using 10 μL of sample and 90 μL DEB in Eppendorf tubes.

9. Measurement of DNA qPCR

Detection of DNA was performed by preparing qPCR mix. (see the “qPCR” protocol in the General Methods). Primers were for the Ng porA gene in each amplification. Samples were quantified to obtain a Cq for concentration estimation before the samples were run in digital PCR (see step 10)

10. Measurements of DNA by ddPCR

Detection of DNA was performed ddPCR following the “ddPCR” protocol in General Methods. Use the Cq obtained from qPCR in step 9 to determine the dilution before quantification with droplet digital PCR (ddPCR). Primers were for the Ng 16S gene

11. Digital PCR Analysis

The ddPCR concentrations reported are corrected for dilutions prior to any CT ratio calculation. The CT ratio is calculated by dividing the DNA concentration in copies/μL of the untreated (control) sample by the treated sample. The ddPCR raw concentration values in copies of target molecule/μL are not shown, but the ratio of these concentrations is shown as the CT ratio in FIG. 7A, FIG. 7B, and FIG. 7C. A CT ratio >1 can occur when the denominator (treated sample concentration) is less than the numerator (untreated/control concentration). A smaller detected concentration in the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids, as a result of the antibiotic exposure, enhancing treatment, and degradation of nucleic acids with a molecule such as DNaseI.

The results illustrated in FIG. 7 show the generality of DNA accessibility method with one-step incubation with an enhancer, since differentiable CT ratios between susceptible and resistant isolates are obtained through this exposure

Digital PCR use for NA amplification and to obtain DNA concentrations here is performed to compare the untreated and treated samples (shown above in the results section as a CT ratio, dividing the concentrations of nucleic acid in one sample by the other). This metric is enough to differentiate between the isolates screened.

The exemplary results of FIG. 7, support the conclusion that Antibiotic concentrations greater than the clinical MIC breakpoint will be used for exposures. This is the case for each resistant isolate above; since the CT ratios for the resistant isolates in each antibiotic screen are not significantly greater than 1.0, this concentration does not seem to add background signal in the resistant isolates at these antibiotic incubation timescales (15 minute exposure).

Example 5: AST Performed by Detecting Inaccessible RNA Quantified with ddPCR, LAMP

Antibiotic susceptibility test was performed with methods herein described wherein detection of inaccessible RNA is performed in an AST comprising adding an RNA degrading enzyme to a sample before cell lysis and detection of RNA in the sample.

In particular this example shows a detection of a difference in amounts of DNA in a treated incubation for an antibiotic-susceptible clinical isolate with respect to a corresponding control, but not for a antibiotic-resistant clinical isolate. The isolates used in this example were ng_3 and ng_30 (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

In particular, in this example detection of RNA was performed in outcome of the following procedure.

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters of media were prewarmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods) for ≥30 minutes prior to use.

For each isolate, two incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the chart below, titled “Antibiotic Exposure per 500 μL”. Add Triton X (enhancer), RNase cocktail (Catalog AM2286) (degrader), NaHCO₃ (conditioner). Antibiotic was added when bacteria cells are incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 3 of this example).

Antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks: Diluted 1 mg/mL ceftriaxone stock, was prepared according to “Preparing Antibiotic Stocks” description in the general methods, see ceftriaxone. To Prepare 50× stocks of the antibiotic follow the following dilutions in nuclease-free (NF) water

Antibiotic was added to the “treated” sample Nuclease free water was added to the untreated sample.

Table of components for media preparation and antibiotic Exposure per 500 μL. (2 mL purple cap tubes setup) Component Volume MH (Prewarmed) 375 μL Triton X-100 detergent (10X, 10%) (enhancer) 50 μL (1X, 1%)   RNase Cocktail (50X) (degrader) 1 μL (0.1X)    Antibiotic (50X), see ABX dilutions above (treated) 10 μL (1X)     (OR replace with NF Water in controls, (untreated) NaHCO₃ (100 mM) 5 μL (1 mM)    Ng Suspension (OD = 0.5) 50 μL (OD = 0.05)

2. Providing a Sample Containing Bacteria

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods

While cell pellet was suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared.

Right before measuring OD, the tubes were set on the shaking heat block (set to 38° C. and 1000 rpm shaking).

3. Optionally, Pre-Process the Sample Containing Bacteria

OD600 (dilute 1/10 in media) was measure and the, OD's below are corrected for the 1/10 dilution for the measurement. ng_30 OD=1.1 (before dilution). Adjust OD of suspension to OD=0.5 in prewarmed MHB+5 mM NaHCO₃ media

4. Split the Sample into Two or More Parts

50 uL of the Ng suspension were withdrawn into a pipette

5. Combine Sample with Incubation Media

50 uL of the Ng suspension were added into “treated” media, and 50 uL of the Ng suspension were added into untreated media. Note this addition can be performed sequentially or in parallel (using multichannel pipet) as will be understood by a skilled person

6. Incubate the treated and untreated samples at a controlled temperature for a short amount of time

Incubate at 37 C for 15 minutes

7. Extraction (at 15 Minutes) of Treated and Untreated Samples to Purify RNA and DNA

Extraction was performed following the procedure outlined herein for “Zymo Column Extraction (RNA)” in the General Methods. Eluted solution is referred to as “extraction”

8. Optionally Splitting the Extractions

In a variation of the experiments exemplified herein, the extractions can be split

-   -   Splitting of the eluted nucelases into two parts. One part gets         treated with DNaseI to remove the DNA (step 9). One part retains         all extracted nucleic acids (RNA+DNA) without any further         processing.

9. Optionally Add Degraders to a Part of the Extraction: DNase Treatment of (an Aliquot) of Extractions to Remove DNA

In a variation of the experiments exemplified herein, a DNase treatment (100 U/mL final concentration of NEB DNaseI) of an aliquot of the Zymo extracted nucleic acids can be performed to obtain a sample of RNA. In particular the DNAase treatment can be performed according to the following procedure.

-   -   10 min at 37° C. (actual incubation closer to 15 minutes—static         incubation in 1.5 mL Eppendorf tubes on benchtop heating block         set to 37° C.)         -   a. dilute DNase to 1000 U/mL (10×)             -   5 μL DNaseI+5 μL NF water         -   add 1 μL of 1000 U/mL DNaseI to each prepared tube

10. Measurement of RNA and DNA by qRT-PCR

Detection of RNA and DNA was performed by preparing a RT-qPCR mix and qPCR mix for the (−RT) controls. (see the “qRT-PCR” protocol in the General Methods). Primers were for the Ng 16S gene in each amplification. Samples were quantified for RNA (+RT), DNaseI-treated aliquots

11. Measurements of RNA and DNA by ddRT-PCR

Detection of RNA was performed according to the “ddRT-PCR” protocol in General Methods, wherein primers were for the Ng 16S gene in each amplification, samples were quantified for total nucleic acid (+RT) and samples quantified for background DNA that was not digested (−RT)

12. Digital PCR Analysis

Digital PCR analysis was performed with the ddRT-PCR concentrations reported corrected for dilutions prior to amplification. CT ratios were computed by dividing the concentrations measured with digital PCR.

13. qLAMP Analysis

Detection by qLAMP analysis was performed according to the “qLAMP” protocol in General methods wherein the IsoAmp Buffer I was used with no adjustments from the commercial mix were done, this contains Tween, the transcription enzyme was Bst 2.0, the LAMP primers were for the Ng 16S gene and the mixt was 20×ng_16 s_1 primer mix=P228/229 (4 uM), P230/231 (32 uM), P327/328 (8 uM). The RT-LAMP was run at 55° C. for 10 minutes and 68° C. for 20 seconds.

The results illustrated in FIG. 8, shows that accessible RNAs are degraded by the added RNases and RNases are inactivated during extraction with the Zymo Research Viral DNA/RNA kit. Due to the treatment with the lysis agent and antibiotics, the Susceptible-treated sample will have a lot of RNA degraded by the RNases, and shows a much lower concentration than the control. There is a small effect seen in the resistant-treated sample as well, but the effect is not as dramatic. Time to positive measurements are shown (10 minutes+TTP at 68° C.)

Comparisons of the RNA concentrations can be shown with the CT ratio, as illustrated in FIG. 9 which shows the CT ratio of the RNA concentrations of the antibiotic-susceptible and antibiotic-resistant Ng isolates. Primers target NG 16S rRNA that has been reverse-transcribed. The ratio is calculated by dividing the concentrations obtained by digital PCR shown in the plot above.

The quantification of RNA through RT-LAMP with Ng 16S rRNA primers is illustrated in FIG. 10. In particular the illustration of FIG. 10, shows that RNA accessibility can show very large differences in nucleic acid concentrations that can be amplified quickly with LAMP. Error bars represent 95% confidence intervals from the qLAMP replicates. Time to positive measurements are shown (10 minutes+TTP at 68° C.), so a TTP of 3 minutes in the plot above, represents 13 minutes since the samples were mixed and added to the thermocylcer. The TTP difference between the treated and untreated samples can be used to differentiate susceptible and resistant isolates. An increase in the TTP of the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids

The results of this example support the conclusion that RNA accessibility can be used for AST via adding an RNA degrading enzyme during the antibiotic incubation that was performed in the presence of an enhancing treatment that retains cell viability, such as 1% Triton X-100 m to then detect the inaccessible RNA following cell lysis.

Digital RNA amplification can used for analysis and the CT ratio of computed from the RNA copies/μL can be used to differentiate between the susceptible and resistant isolates. In the CT ratio plotted in the results section, it suggests that this result can appear binary, the CT ratio is very large or it is around 1.0.

qLAMP amplification on extracted RNA can be used for analysis and the TTP differences could be used to differentiate between antibiotic susceptible and antibiotic resistant isolates. Amplification with bulk LAMP measurements allows for fast readouts.

It is noted that a skilled person will understand upon reading of the present disclosure step 1 can be performed before or after steps 2-4 of this example and that steps 1-7 (or 1-11) can be done on different days for each isolate as long as the comparisons of untreated and antibiotic-treated for one isolate are handled on the same day. It is also noted that step 13 of this example can be performed independently from steps 10-12 as will be understood by a skilled person upon reading of the present disclosure.

Example 6: AST Performed by Detecting Inaccessible RNA, with 1-Step Incubation (Media with Enhancer; Followed by Cell Lysis and NA Extraction) with RNA Quantified with Digital PCR

Antibiotic susceptibility test was performed with methods herein described comprising one-step incubation of a sample with an enhancer with adding an RNA degrading enzyme

The procedure used in this example is identical to the one of Example 5, up until the nucleic acid quantification which in this example is performed according to the following procedures.

Measurement of RNA and DNA by qRT-PCR

Measurement was performed by RT-qPCR mix and qPCR mix for the (−RT) controls. (see the “qRT-PCR” protocol in the General Methods). The primers were for the Ng 16S gene in each amplification. Samples quantified for total nucleic acid (+RT). The samples were quantified for RNA (+RT), DNaseI-treated aliquots. The samples quantified for DNA (−RT)

Measurements of RNA and DNA by ddRT-PCR

Measurement of RNA and DNA was performed following the “ddRT-PCR” protocol in General Methods. Primers were for the Ng 16S gene in each amplification. Samples were quantified for total nucleic acid (+RT) Samples quantified for DNA (−RT)

Digital PCR Analysis

Detection with digital PCR was performed with the ddPCR and ddRT-PCR concentrations reported are corrected for dilutions prior to amplification. For the RNA/DNA ratio the RNA concentrations were obtained by subtracting the concentration of DNA (extractions, without DNaseI treatment, quantified in ddPCR) from the total nucleic acid (extractions, without DNaseI treatment, quantified in ddRT-PCR in the presence of a reverse-transcriptase enzyme to amplify both DNA and RNA).

The results illustrated in FIGS. 11A to 11C show the total NA concentration, (NA=DNA+RNA) measured from the extraction (FIG. 11A) the total DNA concentration, middle (FIG. 11B). as well the C:T ratio computed from the total nucleic acid measurement (ddRT-PCR of extractions in the presence of a reverse-transcription enzyme) (FIG. 11C). The concentrations are quantified in digital PCR, see methods steps 11-12. The RNA concentration can be found by subtracting the DNA concentration from the total NA concentration. In the illustration of FIGS. 11A to 11C the RNA and DNA concentrations are used for the comparison. The DNA concentration is about the same for all four conditions since there is no extracellular DNase added to the tube, the DNA recovered is from both lysed and intact cells. However, the addition of RNase degrades any accessible RNAs.

The RNA concentrations computed from the subtraction of the DNA from the total NA (as described above) are plotted in FIG. 12A. These concentrations can also be used to calculate a CT ratio (FIG. 12B) to differentiate between the susceptible and resistant isolates, giving a very similar readout to the CT ratio computed from the total RNA measurement and conclusions made as with previous DNA accessibility CT ratios. An RNA CT ratio was used since the degrader in the experimental setup includes RNases.

The plot of FIG. 13 shows the ratio of the measured DNA concentrations divided by the computed RNA concentration (total NA concentration minus DNA concentration). The results illustrated in FIG. 13 shows that with one nucleic extraction a ratio can be computed to visualize the change in RNA concentration with respect to the unchanging DNA concentration. In a control case, it is expected for the RNA concentration to be much higher than the DNA concentration. In the case of the susceptible-treated, the RNA concentration is dropping due to the change in RNA accessibility and the presence of the RNases.

In general, the results of the experiments of this example support the conclusion that RNA accessibility can be used for AST via adding an RNA degrading enzyme during a 15-minute antibiotic incubation that can be performed in the presence of an enhancer, such as 1% Triton X-100. Total nucleic acid, total RNA+DNA that is extracted with the Zymo Research Viral DNA/RNA column can be used to generate a CT ratio to correctly characterize CRO susceptibility after 15 minutes of CRO exposure, shown in FIG. 11C.

The results of the experiments of this example support the conclusion that calculating RNA/DNA normalization may be used for AST without requiring splitting of the sample containing bacteria, and without requiring a control incubation. This is confirmed by plot titled “DNA/RNA Ratio+CRO) (bottom). The control DNA/RNA ratios are plotted but are not needed to make the susceptibility call. Only the (+CRO) bars are needed, shown in FIG. 13.

The results of the experiments of this example further support the conclusions that digital NA amplification can be used for this analysis.

Example 7: AST Performed by Detecting Inaccessible DNA with qPCR and Static Incubation

Antibiotic susceptibility test was performed with methods herein described relying on b-lactam-induced cellular lysis, or cell-wall damage, to generate differences in DNA accessibility between the unexposed and exposed tubes. No enhancing treatment is performed in this example. In this example DNaseI is used to degrade the DNA made accessible by beta-lactam induced damage over incubation times larger than what is tested in examples 1-6 (incubation times >30 minutes)

The layout of this experiment is illustrated in FIG. 14 according to which a penicillin-susceptible isolate is incubated with penicillin for 15 min-6 hours. At each time point, DNaseI will be added to degrade the accessible DNA, then the DNaseI will be inactivated simultaneously with the lysis of the remaining cells. The quantified nucleic acids represent those from the cells with inaccessible DNA throughout the antibiotic exposure and incubation with DNaseI.

In particular, the detection was performed in outcome of the following procedure.

-   -   1. Provide Incubation Media Containing Enhancer, Accessibility,         with and without an Antibiotic for Each Isolate

Several milliliters of media were prewarmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods) for ≥45 minutes prior to use.

For each isolate, two incubation tubes were prepared in 12 mL BD Falcon culture tubes, following the chart below, titled “Antibiotic Exposure per 2 mL”. Add NaHCO₃ (conditioner). Antibiotic was added when bacteria cells were incubating, so that the time from thawing the antibiotic stock was minimized before exposure (see step 4 of this example).

Primary Incubation: Antibiotic Exposure per 2 mL. (12 mL BD Falcon culture tubes) Component Volume MHB (Prewarmed) 1,770 μL Antibiotic (50X), see ABX dilutions above (treated) 40 μL (1X) (OR replace with NF Water in controls, (untreated) NaHCO₃ (100 mM) 90 μL (4.5 mM) Ng Suspension (OD = 1) 100 μL (OD = 0.05, 0.5 mM NaHCO₃)

Secondary Incubation: DNAaseI Degradation Exposure per 50 μL. (200 μL capacity PCR tubes) Component Volume MHB (Prewarmed) 22.5 μL 10X NEB DNaseI Reaction Buffer 5 μL (1X) NEB DNaseI (2000 U/mL) 2.5 μL (100 U/mL) Ng sample from primary incubation 20 μL

2. Providing a Sample Containing Bacteria

The isolate used was ng_15, a penicillin-susceptible isolate with an MIC of 0.015 μg/mL penicillin. (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section. While cell pellet was suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared. Right before measuring OD, the tubes were set on the shaking block in the 37° C., 5% CO₂ incubator.

3. Optionally, Pre-Process the Sample Containing Bacteria

In a variation of the procedure exemplified herein, the OD600 (dilute 1/10 in media) was measured and the OD of suspension to OD=0.5 in pre-warmed MHB+5 mM NaHCO₃ media was adjusted.

4. Add the Antibiotic

Antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks as follows: diluted 1 mg/mL ceftriaxone stock, were prepared according to “Preparing Antibiotic Stocks” description in the general methods, see penicillin.

In particular, 1 mg/mL penicillin stock was diluted to make a 50× stock for the experimental exposure. The 1 mg/mL stock can be stored at −80° C. in single-use aliquots prepared from penicillin G sodium salt. In particular 500 μL of 50× (1×1 μg/mL PEN→50 μg/mL=0.050 mg/mL PEN) penicillin were prepared with 25 μL (1.0 mg/mL stock)+475 μL nuclease-free water.

The antibiotic was added to the corresponding exposure tubes setup in step 1 of the present example.

5. Split the Sample into Two or More Parts

100 uL of the Ng suspension were withdrawn into a pipette (following the chart for the primary exposure in step 1 of the present example).

6. Combine Sample with Incubation Media

The timer was started and the Ng suspension were added to the sample. The time was recorded for Lab Notebook records. 100 uL of the Ng suspension were added into “treated” media, add 100 uL of the Ng suspension into untreated media. This addition can be performed sequentially or in parallel (using multichannel pipet) as will be understood by a skilled person.

Before transporting the tubes to the incubator, it was proceeded to steps 8-9 to take a “t0” sample. Tubes were incubated on the shaker (500 rpm) in 37° C., 5% CO₂ incubator for the duration of the experiment.

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for Long Amount of Time (6 Hours) Taking Aliquots for Analysis, See Steps 7-8, at Defined Time Intervals

The treated and untreated samples were incubated at 37 C for 15 minutes, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr. At each time point, bring the culture tubes into the BSC and incubate an aliquot of the contents with DNaseI to degrade the accessible DNA.

8. Secondary Incubation of Treated and Untreated Samples to Degrade Accessible DNA

20 uL of the sample were withdrawn from the primary exposure into a pipette and transferred to the secondary incubation for accessible DNA degradation in a PCR tube. The caps were closed tightly, and the tubes were vortexed for a few seconds and centrifuged on a benchtop centrifuge to remove droplets from the caps.

These tubes were incubated for 5 min. incubation at 37° C. on a thermocylcer with a heated lid. The lid of the thermocylcer was lifted at 4 min to let the caps cool and bring tubes back into the BSC, vortex briefly, centrifuge

9. DNA Extraction of Treated and Untreated Samples from the Secondary Incubation, at Each Time Point of Primary Exposure (at 0, 15 Minutes; 1, 2, 3, 4, 5, 6 Hours) to Purify DNA

DNA extraction was performed following the procedure outlined in “DEB Extraction” in the general methods section, using 10 μL of sample and 90 μL DEB in Eppendorf tubes. The extraction was stored at −20° C. until all time points were collected. The operator proceeded to step 10 to measure DNA from each time point.

10. Measurement of DNA by qPCR

DNA was measure by preparing a qPCR mix (see the “qPCR” protocol in the General Methods). The Primers were for the Ng 16S gene in each amplification, the samples were run in triplicate for 35 cycles at an annealing temperature of 62° C. Cqs measurements were used to calculate the CT ratio at each time point

The results illustrated in FIGS. 15A to 15 C show that by relying on beta-lactam induced changes in accessibility (such as lysis or disruption of the cell envelope) without the addition of an enhancer or additional reagent (other than media, cells, antibiotic, and DNaseI) become detectable after several hours of exposure to the antibiotic. These figures show how the CTR magnitude changes with antibiotic exposure times. For the purposes of using this method to determine susceptibility, only one time would be needed. Based on these data, one isolate would require two DNA extractions, a control and a treated extraction ≥3 hours.

The exemplary results illustrated in FIGS. 15A to 15 C support the conclusion that DNA accessibility with a degradation step (inclusion of DNaseI) help highlight the change in DNA accessibility caused by cell wall damage from the beta-lactam incubation; increase in Cq in the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids. MHB is not a media that sustains cell growth of NG over these time scales, supporting the conclusion that nucleic acids are made accessible by cell wall damage. DNaseI can be treated as a secondary step, but it could be included for the duration of the antibiotic exposure instead.

It is noted that step 1 of this example can be performed before or after steps 2-3 of this example, or in parallel with step 4 of this example as will be understood by a skilled person.

Example 8: AST Performed by Detecting Inaccessible DNA with Mechanical Disruption, after ABX Exposure Before Cell Lysis and NA Extraction

Antibiotic susceptibility test was performed with methods herein described in which mechanical disruption by sonication is used as an enhancer for beta-lactam incubation, in combination with DNaseI as a degrader.

The layout of the example is illustrated in FIG. 16 which provides a schematic representation of the experimental steps.

In particular, the detection was performed in outcome of to the following procedure

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters of media were prewarmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods) for ≥45 minutes prior to use.

For each isolate, two incubation tubes were prepared in 2 mL purple cap tubes (VWR Cat #89004-298), following the chart below, titled “Primary Incubation (Antibiotic Exposure)”. Add NaHCO₃ (conditioner). Antibiotic were added when bacteria cells were incubating, so that the time from thawing the antibiotic stock was minimized before exposure (see step 4 of this example).

Primary Incubation (Antibiotic Exposure): (500 μL in screw-cap tube) Component Volume Ng Suspension (OD = 0.5) in MHB + 5 mM 50 μL NaHCO₃ (OD = 0.05, 0.5 mM NaHCO₃) MHB 417.5 μL NaHCO₃ (100 mM) 22.5 μL (4.5 mM) 50X ABX or NF water in control 10 μL

For each isolate, two incubation tubes were prepared of each secondary incubation. Each sample was added to a tube of each option so that the data can be compared with and without the second enhancer of the surfactant).

Secondary Incubation (option 1): DNaseI digestion after sonication. (50 μL in PCR tube) Component Volume Ng Suspension (Post-Covaris Sonication) 20 μL NEB DNaseI (2000 U/mL) 2.5 μL (100 U/mL) 10X DNaseI Reaction Buffer 5 μL MHB 22.5 μL

Secondary Incubation (option 2): DNaseI digestion in the presence of enhancer after sonication. (50 μL in PCR tube) Component Volume Ng Suspension (Post-Covaris Sonication) 20 μL NEB DNaseI (2000 U/mL) 2.5 μL (100 U/mL) 10X DNaseI Reaction Buffer 5 μL MHB 20 μL TNP (100 mM, 20X) 2.5 μL (5 mM, 1X)

2. Providing a Sample Containing Bacteria

ng_17 is a penicillin-susceptible isolates and ng_15 is a ceftriaxone-susceptible isolate were used. These isolates were exposed to an antibiotic concentration of 1.0 μg/mL penicillin or 4.0 μg/mL ceftriaxone (treated) and to nuclease-free water (control). (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section. While cell pellet was suspending, the antibiotic exposure condition and the secondary incubations in PCR tubes (everything except Ng suspension and ABX) was prepared as outlined in step 1 of this example.

Right before measuring OD, these tubes were set on the shaking block in the shaking heat block for incubation at 37° C.

3. Optionally, Pre-Process the Sample Containing Bacteria

In a variation of the experimental procedure herein described the OD600 (dilute 1/10 in media) was measured and the OD of suspension was adjusted to OD=0.5 in pre-warmed MHB+5 mM NaHCO₃ media.

4. Add the Antibiotic

Antibiotic dilutions were prepared from pre-made and frozen antibiotic stocks as follows. Diluted 1 mg/mL ceftriaxone or penicillin stock, was prepared according to “Preparing Antibiotic Stocks” description in the general methods, see penicillin and ceftriaxone.

In particular, 1 mg/mL penicillin stock was diluted to make a 50× stock for the experimental exposure. The 1 mg/mL stock was stored at −80° C. in single-use aliquots prepared from penicillin G sodium salt. In particular 500 μL of 50× (1×1 μg/mL PEN→50 μg/mL=0.050 mg/mL PEN) penicillin were prepared with 25 μL (1.0 mg/mL stock)+475 μL nuclease-free water.

1.0 mg/mL ceftriaxone stock was diluted, from −80° C. storage. In particular, 50× Stock (1×4 μg/mL CRO→200 μg/mL=0.200 mg/mL CRO) 250 μL of 0.200 mg/mL=50 μL (1.000 mg/mL stock)+200 μL NF water.

The antibiotic was added to the corresponding exposure tubes setup in step 1 of this example.

5. Split the Sample into Two or More Parts

50 uL of the Ng suspension were withdrawn into a pipette (following the chart for the primary exposure in step 1 of this example)

6. Combine Sample with Incubation Media

The timer was started and the Ng suspension was added. The time was recorded for the Lab Notebook records. 50 uL of the Ng suspension were added into “treated” media, and 100 uL of the Ng suspension were added into untreated media. This addition can be performed sequentially or in parallel (using multichannel pipet) as will be understood by a skilled person.

The tubes were staggered so that timing was more precise for each of and to give some timing for sonication, plating, and extractions of the tubes. The timer was started and the Ng suspension was added. The time was recorded for the Lab Notebook records. (at OD600=0.5 suspension, tubes were incubated on the shaker (1000 rpm) at 38° C. on the heat-shake block between the staggered exposure tubes).

7. Incubate for a Short Time (15 Minutes) with Antibiotics

The exposure tubes were incubated for 15-minutes on the heat-shake block set to 1000 rpm and 38° C.

8. Mechanical Disruption by Sonication

For each exposure tub, a 50 μL aliquot of the exposure tube was taken into a Covaris microTUBE-50. In cases like the one below, a separate 50 μL aliquot was taken for each sonication parameter. The chart below of Covaris Sonicating Conditions was followed to select the conditions for each sample. All samples were maintained at room temperature for the duration of the handling of the samples in parallel

These steps were performed separately for the untreated and treated

Covaris M220 Sonicating Conditions Peak Incident Duty Cycles Sample power Factor per Duration Group (W) (%) burst (s) Temperature 1-2 N/A N/A N/A N/A N/A 3-4 15 10 200 30 37° C. 5-6 3 10 200 30 37° C.

9. Secondary Incubation of Treated and Untreated Samples

20 uL of the sample were withdrawn from the primary exposure into a pipette and transfer to the secondary incubation for accessible DNA degradation in a PCR tube. Optionally, the sample prepared in step 8 can be divided into multiple secondary incubations (e.g. the two options prepared in step 1 of this example). The caps were closed tightly, the tubes were vortexed for a few seconds and centrifuged on a benchtop to remove droplets from the caps. These tubes were incubated for 5 min at 37° C. on a thermocylcer with a heated lid. The lid of the thermocylcer was lifted at 4 min to let the caps cool. The tubes were brought back into the BSC, vortex briefly, centrifuge

10. DNA Extraction of Treated and Untreated Samples from the Secondary Incubation to Purify DNA

DNA extraction was performed following the procedure outlined in “DEB Extraction” in the general methods section, using 10 μL of sample and 90 μL DEB in PCR tubes using the thermocylcer for heating steps. Since there were many samples that occur in parallel, a separate heat block was used for the DNaseI digestion steps in step 9 of this example as the DEB extraction steps in step 10.

11. Measurement of DNA by qPCR

Measurement of DNA was performed by preparing a qPCR mix (see the “qPCR” protocol in the General Methods). Primers were for the Ng 16S gene in each amplification. Samples were run in triplicate for 35 cycles at an annealing temperature of 62° C.

Cqs measurements were used to calculate the CT ratio at each time point

The results illustrated in FIGS. 17A and 17B, show that some sonication conditions allow for an enhancement of the accessibility of DNA in the susceptible-treated sample compared to the susceptible-control sample. In particular the results in the FIG. 17B labeled with the CTR points show that the readout of CTR depends on the sonication settings (here represented by the Peak Power of sonication in watts [W]).

A skilled person would understand that only one of these conditions screened could be used to determine the susceptibility to penicillin. This single point is obtained from steps (1-7, for step 8 only the third row of the “Covaris M220 Sonication Conditions” is needed to test, since the first two rows did not show a CT ratio >1, and steps 10-11).

The results illustrated in FIGS. 18A to 18 C show sonication conditions that allow for enhancement of accessibility of DNA. These figures differ from FIGS. 17A and 17B in that the related results is a different antibiotic and a different isolate. Conditions might vary between antibiotics. The illustration of FIGS. 18A to 18C with isolate ng_15 and ceftriaxone shows little change with sonication+DNaseI digestion, but shows detectable signals with the combination of the enhancer sonication and the enhancer of surfactant TNP in the DNaseI digestion step.

In particular, the results in the FIG. 18C labeled with the CTR points show that the readout of CTR can be enhanced with the combination of sonication and a surfactant in the DNaseI digestion step. There are three potential conditions that could be used to determine the susceptibility to ceftriaxone if the enhancement step (under the conditions tested) happens after the 15 min antibiotic exposure. These conditions include the DNaseI digestion+5 mM TNP digestion with or without sonication.

In particular, for the CTR point at 0 s sonication with “DNaseI+5 mM TNP digestion” the following steps were performed

-   -   Steps 1-7     -   (step 8 is “skipped” since the sonication refers to the first         row of the table “Covaris M220 Sonicating Conditions” which is         left intentionally empty, to indicate this step is skipped)     -   Steps 9-11

For the CTR point at 30 s sonication with “DNaseI+5 mM TNP digestion” the following steps were performed

-   -   Steps 1-7     -   Step 8: with the second row of the table “Covaris M220         Sonicating Conditions”     -   Steps 9-11

For the CTR point at 120 s sonication “DNaseI+5 mM TNP digestion” the following steps were performed

-   -   Steps 1-7     -   Step 8: with the third row of the table “Covaris M220 Sonicating         Conditions”     -   Steps 9-11

The results illustrated in FIGS. 17A to 17 C support the conclusions that mechanical disruption by sonication after 15-minute penicillin exposure is a sufficient enhancer.

The results illustrated in FIGS. 18A to 18C also support the conclusions that mechanical disruption by sonication after 15-minute ceftriaxone exposure is done, followed by incubation with a second enhancer in the DNaseI digestion step (such as a 5-minute incubation with the surfactant TNP, at 5 mM, at 37° C.)

The results in FIGS. 18A to 18C illustrate that the combination of two enhancing treatments can give an even-bigger response, as measured by changes in the detectable inaccessible nucleic acids, which is shown in the figures as larger Cq differences. As with other qPCR representations and analysis, an increase in Cq in the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids.

The results illustrated in FIGS. 17A to 17 C and in FIGS. 18A to 18C further support the conclusions that qPCR is used for DNA measurements and data analysis and CT ratios computed from qPCR Cqs can be used to detect susceptibility. As with other qPCR representations and analysis, an increase in Cq in the antibiotic-treated sample corresponds to decreased quantity of detectable inaccessible nucleic acids

It is noted that step 1 of this example can be performed before or after steps 2-3, of this example or in parallel with step 4 of this example as will be understood by a skilled person.

Example 9: AST Performed by Detecting Inaccessible DNA with Mechanical Disruption, DNA Accessibility AST Sonication During ABX Exposure

Antibiotic susceptibility test was performed with methods herein described in which mechanical disruption by sonication is used as an enhancer for beta-lactam incubation, in combination with DNaseI as a degrader.

The layout of the example is illustrated in FIG. 19 which provides a schematic representation of the experimental steps.

In particular, FIG. 19 is a representation of the experimental steps, in which a ceftriaxone-susceptible isolate is incubated with ceftriaxone and DNaseI for 15 minutes on aCovaris M220 sonicator (with a protocol of 30 s sonication, 30 s holding at 37° C.). From there, there are two options. One, the DNaseI is inactivated at the remaining cells are lysed, followed by qPCR. Two, there is an additional 5 minute incubation in the presence of surfactant (10 mM CHAPS) and DNaseI to degrade the accessible DNA. The DNaseI will be inactivated simultaneously with the lysis of the remaining cells. In both cases, the quantified nucleic acids represent those from the cells with inaccessible DNA throughout the antibiotic exposure, sonication, and incubation with DNaseI.

In particular, the detection was performed according to the following procedure:

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters (at least 14 mL) of media were prewarmed in a 37° C., 5% CO₂ incubator (media is MHB, see media preparation in general methods) for ≥45 minutes prior to use.

For each isolate, two incubation tubes were prepared in screw-cap Covaris microtube-50, following the chart below, titled “Primary Incubation (Antibiotic Exposure)”. NaHCO₃ (conditioner) was added. Wait to add antibiotic until bacteria cells are incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 4 of this example).

Primary Incubation (Antibiotic Exposure): (50 μL in screw-cap Covaris microTUBE) Component Volume Ng Suspension (OD = 1) in MHB + 5 mM 10 μL NaHCO₃ (OD = 0.2, 0.5 mM NaHCO₃) MHB 27.75 μL NaHCO₃ (100 mM) 2.5 μL (4.5 mM) 10X ABX or NF water in control 5 μL NEB DNaseI (2000 U/mL) 5 μL (100 U/mL)

For each isolate, two incubation tubes of each incubation below (each sample gets added to a tube of each option) were prepared. This way the data can be compared with and without the second enhancer of the surfactant).

“t0” Incubation: DNaseI digestion of initial cell suspensions. (50 μL in PCR tube) Component Volume Ng Suspension (Post-Covaris Sonication) 10 μL NEB DNaseI (2000 U/mL) 2.5 μL (100 U/mL) MHB 37.5 μL

In some embodiments, after DEB extraction of 10 μL of the sonicated solution, additional steps are described to be taken to enhance the accessibility to DNA to the degrader (DNaseI) that is already present in the sample suspension. This is achieved by pipetting 2.1 μL of enhancer into the covaris microTUBE and incubating in a glass bead bath, such that the contents of the covaris microTUBE are kept at 37° C.

Step 1 can be performed before or after steps 2-3 or in parallel with step 4. as will be understood by a skilled person upon reading of the present disclosure.

Secondary Incubation: DNaseI digestion in the presence of enhancer after sonication. (42.1 μL in screw-cap Covaris microTUBE) Component Volume Ng Suspension (Post-Covaris 40 μL (100 U/mL DNaseI, Sonication) 1X ABX, 5 mM NaHCO₃) CHAPS (200 mM, 20X) (enhancer) 2.1 μL (10 mM, 1X)

2. Providing a Sample Containing Bacteria

ng_19 is a ceftriaxone-susceptible isolate and ng_30 is a ceftriaxone-reduced-susceptible isolate. (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section with the following changes: incubate cell resuspension on shaker (1000 rpm) in 37° C., 5% CO₂ incubator (≥10 minutes).

The culture tube was then returned to the shaker in the incubator. This protocol requires the control and treated incubations to be staggered by 20 minutes.

While cell pellet is re-suspending, prepare the antibiotic exposure condition (everything except Ng suspension and ABX), see table below, and prewarm the tube in a glass bead bath so that the liquid inside the tube is 37° C. In the case of this lab setup, the bead bath was set to 40° C. and the temperature of an equivalent volume was measured to be 37° C. with a thermocouple.

3. Optionally, Pre-Process the Sample Containing Bacteria

OD600 (dilute 1/10 in media) was measured. OD of suspension was adjusted to OD=1.0 in pre-warmed MHB media+5 mM NaHCO₃

4. Add the Antibiotic

1.0 mg/mL ceftriaxone (CRO) stock was diluted, from −80° C. storage. In particular, 10× Stock was prepared (1×1 μg/mL CRO 10 μg/mL=0.010 mg/mL CRO) in a 500 volume by mixing 5 μL (1.000 mg/mL stock)+495 μL NF water.

The antibiotic was added to the corresponding exposure tubes setup in step 1. Since the exposures are staggered by 20 minutes in this setup, the antibiotic dilution was stored on ice and was added within 10 minutes of beginning the exposure.

5. Split the Sample into Two or More Parts

10 uL of the Ng suspension (from step 3) was withdrawn into a pipette (following the chart for the primary exposure in step 1)

6. Combine Sample with Incubation Media

The time starts and the Ng suspension was added. The time is recorded for Lab Notebook records. 10 uL of the Ng suspension was added into “treated” or “untreated” media; this addition can be performed sequentially or in parallel (using multichannel pipet). The tubes were staggered so that timing can be more precise for each of and to give some timing for sonication, plating, and extractions of the tubes. Start the timer and add the Ng suspension. Record the time for Lab Notebook records. The screw-cap covaris microTUBE were sealed with parafilm.

7. Incubate for a Short Time (15 Minutes) with Antibiotics and Mechanical Disruption by Covaris Sonication

The exposure tubes were incubated for 15-minutes in the covaris sonicator water bath set to 37° C. The sonicator was started to run the following protocol in the table below:

Covaris Sonicating Conditions Peak Duty Cycles Sample Incident Factor per Duration Group power (W) (%) burst (s) Temperature Method Name all 75 2 100 [30 s on, 37° C. Ng_test_713_C 30 s delay]*15

8. While Running Steps 6-7; Run a Parallel t0 Exposure

Cells are added to the t0 tube (as with step 5) and the exposure tubes for the static incubation at the same time, then get vortexed, spun down and placed on the 37° C. heat block Benchmark Multi-Therm set to 37° C. and 0 rpm. Follow the Chart for the incubation tubes prepared in step 1.

9. DNA Extract

DNA extraction was performed following the procedure outlined herein “DEB Extraction” in the general methods section, using 10 μL of sample (of the 50 μL total in the covaris microTUBE) and 90 μL DEB in PCR tubes using the thermocylcer for heating steps. SInce there are many samples that occur in parallel, a separate heat block is used for the DNaseI digestion steps in step 8 and 9 as the DEB extraction steps here in step 10. Additionally, the samples in covaris microTUBEs cannot be vortexed, these steps were replaced by a mixing step by pipetting (pipetting up and down with a P100 set to 20 μL)

10. Secondary Incubation of Samples

At this point 10 μL of the 50 μL has been taken out for the condition without the additional enhancer

-   -   add 2.1 uL of the enhancer to the covaris TUBE with the         remaining 40 μL of sample     -   Mix by pipetting up and down with a P100 set to 20 μL     -   Screw the cap shut and ensure it is sealed with parafilm.     -   Incubate these tubes for 5 min. in the glass bead bath (set to         40° C. but measured to be 37° C. inside these tubes)     -   Return the sample to the BSC for an addition DEB extraction

11. DNA Extraction of Treated and Untreated Samples from the Secondary Incubation to Purify DNA

step 10 was repeated for the tube that incubated with the enhancer.

12. Repeat Steps 3-11 for Each Condition of Each Isolate

13. Measurement of DNA by qPCR

qPCR mix is prepared as follows (see the “qPCR” protocol in the General Methods):

-   -   Primers were for the Ng 16S gene in each amplification     -   Samples were run in triplicate     -   35 cycles     -   Annealing temperature of 62° C.

Cqs measurements were used to calculate the CT ratio at each treated, untreated pair at the same conditions.

The results illustrated in FIGS. 20A to 20C summarize three parts for a susceptible isolate. The left-most panel shows the t0 qPCR measurements for the control (untreated) and the treated tubes; these qPCR measurements are used to normalize the AST measurements in the middle panel, since the control and treated incubations in the middle panel were taken 20 minutes apart. Four measurements are used in this example to compute a single t0-normalized CTR point.

The result in the third panel labeled “[sonication] 4 DEB” can be used to determine susceptibility to ceftriaxone. This point is obtained from steps (1-9, and 13). The result in the third panel labeled “[sonication]+5 min. CHAPS” is obtained from steps (1-8, and 10-13).

The figure above summarizes three parts for the resistant isolate. The left-most panel shows the t0 qPCR measurements for the control (untreated) and the treated tubes; these qPCR measurements are used to normalize the AST measurements in the middle panel, since the control and treated incubations in the middle panel were taken 20 minutes apart. Four measurements are used in this example to compute a single t0-normalized CTR point.

The result in the third panel labeled “[sonication] 4 DEB” can be used to determine susceptibility to ceftriaxone. This point is obtained from steps (1-9, and 13). In this case, the CTR close to 1.0 is indicative of no extra change from the treated condition, and this result shows the resistant phenotype.

The result in the third panel labeled “[sonication]+5 min. CHAPS” is obtained from steps (1-8, and 10-13). As with the first data point in this panel, the CTR close to 1.0 is indicative of no additional change in accessibility from the treated condition, and this result shows the resistant phenotype.

The results shown in FIGS. 20A to 20C supports the conclusions that a differentiable CTR can be resolved with or without the surfactant digestion step when comparing the susceptible and resistant isolates.

In some embodiments, a CTR computed from the treated and untreated samples after a 15 minute antibiotic exposure with sonication can be sufficient to differentiate between the ceftriaxone-susceptible and ceftriaxone-resistant isolates shown in the above plots

In some embodiments, a CTR computed from the treated and untreated samples after a 15 minute antibiotic exposure with sonication, followed by an additional incubation with another enhancer, can be used to differentiate between the ceftriaxone-susceptible and ceftriaxone-resistant isolates shown in the above plots

In some embodiments, qPCR is used for DNA measurements and data analysis and CT ratios computed from qPCR Cqs can be used to detect susceptibility.

Example 10: Susceptibility Ratio Compared to Fold Change Before and after Incubation with ABX (Mechanical aAST Setup)

Antibiotic susceptibility test was performed with methods herein described in which mechanical disruption by sonication is used as an enhancer for beta-lactam incubation, in combination with DNaseI as a degrader.

The workflow of the example is illustrated in FIG. 21, which is the same as in Example 9, but the data analysis is slightly modified. This example only requires the “antibiotic-treated” tube and compared the fold change increase in DNA accessibility from before and after the antibiotic exposure.

DNaseI is used to degrade the DNA made accessible by the combination of beta-lactam induced damage and mechanical disruption by sonication. Cell suspensions are exposed to beta lactams for 15 minutes with 30-second pulses of sonication to selectively enhance the amount of DNA accessible in the susceptible-treated sample.

In some cases, and additional incubation step with a surfactant, such as CHAPS, are used to further enhance this signal.

DNaseI is inactivated during the DEB extraction and the nucleic acids of the cells that remained intact through the exposure protocol are quantified.

Isolates tested are: ng_19 and ng_30. ng_19 is a ceftriaxone-susceptible and penicillin-resistant isolate and ng_30 is a ceftriaxone reduced-susceptible isolate. Both exposed to an antibiotic concentration of 1.0 μg/mL ceftriaxone (treated).

In particular, as shown in FIG. 21, a ceftriaxone-susceptible isolate is incubated with ceftriaxone and DNaseI for 15 minutes on aCovaris M220 sonicator (with a protocol of 30 s sonication, 30 s holding at 37° C.). From there there are two options. One, the DNaseI is inactivated at the remaining cells are lysed, followed by qPCR. Two, there is an additional 5 minute incubation in the presence of surfactant (10 mM CHAPS) and DNaseI to degrade the accessible DNA. The DNaseI will be inactivated simultaneously with the lysis of the remaining cells. In both cases, the quantified nucleic acids represent those from the cells with inaccessible DNA throughout the antibiotic exposure, sonication, and incubation with DNaseI.

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters (at least 14 mL) of media were prewarmed in a 37° C., 5% CO₂ incubator. The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section with the following changes: (media is MHB with 5 mM NaHCO₃, or GW media, see media preparation in general methods) for ≥45 minutes prior to use.

For each isolate, two incubation tubes screw-cap Covaris microtube-50 were prepared, following the chart below, titled “Primary Incubation (Antibiotic Exposure)”. Add NaHCO₃ (conditioner). Wait to add antibiotic until bacteria cells are incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 4 in this example).

Primary Incubation (Antibiotic Exposure): (50 μL in screw-cap Covaris microTUBE) Component Volume Ng Suspension (OD = 1) in MHB + 5 mM 10 μL NaHCO₃ (OD = 0.2, 0.5 mM NaHCO₃) MHB (media) 27.75 μL NaHCO₃ (100 mM) 2.5 μL (4.5 mM) 10X ABX 5 μL (1X) NEB DNaseI (2000 U/mL) (degrader) 5 μL (100 U/mL)

For each isolate, one incubation tubes of the “t0” condition described below was prepared

“t0” Incubation: DNaseI digestion of initial cell suspensions. (50 μL in PCR tube) Component Volume Ng Suspension (Post-Covaris Sonication) 10 μL NEB DNaseI (2000 U/mL) 2.5 μL (100 U/mL) MHB 37.5 μL

2. Providing a Sample Containing Bacteria

ng_19 is a ceftriaxone-susceptible/penicillin-resistant isolate and ng_30 is a ceftriaxone-reduced-susceptible isolate. (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

Cell pellets were resuspended. Cell pellet was scraped with inoculating loop and twirl in 2 mL media (pre-warmed MHB+5 mM NaHCO₃ or GWM), and then incubated on shaker (1000 rpm) in 37° C., 5% CO₂ incubator (≥10 minutes).

While cell pellet is re-suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared. The tube was prewarmed in a glass bead bath so that the liquid inside the tube is 37° C. In the case of this lab setup, the bead bath was set to 40° C. and the temperature of an equivalent volume was measured to be 37° C. with a thermocouple.

3. Optionally, Pre-Process the Sample Containing Bacteria

OD600 (dilute 1/10 in media) was measured. OD of suspension was adjusted to OD=1.0 in pre-warmed MHB media+5 mM NaHCO₃

4. Add the Antibiotic

The antibiotic dilutions are prepared below:

-   -   Ceftriaxone (CRO): Diluted 1.0 mg/mL ceftriaxone stock, from         −80° C. storage. 10× Stock prepared (1×1 μg/mL CRO 10         μg/mL=0.010 mg/mL CRO) in a 500 μL volume by mixing 5 μL (1.000         mg/mL stock)+495 μL NF water     -   Penicillin (PEN): Diluted 1.0 mg/mL penicillin stock, from         −80° C. storage. 10× Stock prepared (1×1 μg/mL PEN→10         μg/mL=0.010 mg/mL CRO) in a 500 μL volume by mixing 5 μL (1.000         mg/mL stock)+495 μL NF water

The antibiotic was added to the corresponding exposure tubes setup in step 1

5. Split the Sample into Two or More Parts (Steps 6-7 and Step 8 Represent this Split into Two Parts)

10 uL of the Ng suspension (from step 3) was withdrawn into a pipette (following the chart for the primary exposure in step 1)

In this example, the sample will be split into a “t0” incubation and a “treated” incubation in step 6.

6. Combine Sample with Incubation Media

The timer started and 10 uL of the Ng suspension was added into “treated” media of the Ng suspension. The time was recorded for Lab Notebook records.

The screw-cap covaris microTUBE was sealed with parafilm and the tube was transported to the covaris sonication (step 7).

7. Incubate for a Short Time (15 Minutes) with Antibiotics and Mechanical Disruption by Covaris Sonication

The exposure tubes were incubated for 15-minutes in the covaris sonicator water bath set to 37° C. The sonicator was started to run the following protocol in the table below:

Covaris Sonicating Conditions Peak Incident Duty Cycles Sample power Factor per Duration Group (W) (%) burst (s) Temperature all 75 2 100 [30 s on, 37° C. 30 s delay]*15

8. While Running Steps 6-7; Run a Parallel t0 Exposure

Cells are added to the t0 tube (as with step 5) and the exposure tubes for the static incubation at the same time, then get vortexed, spun down and placed on the 37° C. heat block Benchmark Multi-Therm set to 37° C. and 0 rpm. Follow the Chart for the incubation tubes prepared in step 1.

9. DNA Extract

DNA extraction was performed following the procedure outlined herein “DEB Extraction” in the general methods section, using 10 μL of sample (of the 50 μL total in the covaris microTUBE) and 90 μL DEB in PCR tubes using the thermocylcer for heating steps. The samples in covaris microTUBEs cannot be vortexed, these steps were replaced by a mixing step by pipetting (pipetting up and down with a P100 set to 20 μL)

10. Measurement of DNA by qPCR

qPCR mix was prepared as follows (see the “qPCR” protocol in the General Methods):

-   -   Primers were for the Ng 16S gene in each amplification     -   Samples were run in triplicate     -   35 cycles     -   Annealing temperature of 62° C.

Cqs measurements were used to calculate the t0/TREATED ratio at each treated, t0 pair for each isolate-antibiotic condition.

The results in FIG. 22 show a possible comparison of two samples to make a susceptibility call. The antibiotic-treated sample undergoes a sonication treatment as described above and the t0 reference undergoes only a DNaseI treatment but no enhancing treatment. The antibiotic-treated samples (the second bar in each pair) represent a sample that has undergone the combination treatment of sonication at 37° C. and incubation with either CRO or PEN antibiotics. In each case the second bar is higher than the initial amount of inaccessible nucleic acids. FIG. 22B shows the conversion of the raw qPCR measurements into a ratio that could be used to predict susceptibility. The t0/TREATED ratio is a representation of how much the accessibility of DNA has changed with the enhancement treatment and the antibiotic treatment. A suggested threshold of susceptibility of a t0/TREATED ratio of 5.0 is represented as a dashed line, in which the susceptible incubations would be expected to fall above this line and the resistant incubations would be expected to fall below this line.

The results illustrated in FIG. 22 supports the conclusion that it is possible to simplify the measurement of susceptibility for the sonication-during-exposure to include only two extractions per measurement (whereas Example 9 required four extractions per measurement) if the t0 and the treated tubes are compared.

Example 11: Two-Step aAST with Surfactant Only in the Secondary Exposure

This example was performed using a two-step exposure in which the first step is exposure to a beta-lactam antibiotic and the second step is a 1:1 dilution into DNaseI treatment in the presence of a surfactant.

The inclusion of the surfactant enhances the accessible nucleic acids made available by short (15 minute) b-lactam exposure by adding a second incubation step with a surfactant. Differences in DNA accessibility between the unexposed and exposed tubes are enhanced. DNaseI is used to degrade the DNA made accessible by beta-lactam treatment and the surfactant.

DNaseI is inactivated during the DEB extraction and the nucleic acids of the cells that remained intact through the exposure protocol are quantified.

ng_15 is a ceftriaxone-susceptible isolate with an MIC≤0.008 μg/mL ceftriaxone. ng_17 is a ceftriaxone-susceptible isolate with an MIC≤0.008 μg/mL ceftriaxone. ng_19 is a ceftriaxone-susceptible isolate with an MIC=0.015 μg/mL ceftriaxone. ng_30 is an isolate with reduced-susceptibility to ceftriaxone with an MIC=0.05 μg/mL ceftriaxone. All isolates are exposed to an antibiotic concentration of 1.0 μg/mL ceftriaxone (treated) and nuclease-free water (control)

FIG. 23 describes the layout of this experiment. An NG isolate is incubated with a beta-lactam antibiotic for 15 minutes, followed by a DNaseI digestion step in the presence of surfactant. At each time point, DNaseI will be added to degrade the accessible DNA, then the DNaseI will be inactivated simultaneously with the lysis of the remaining cells. The quantified nucleic acids represent those inaccessible from the DNaseI throughout the exposure protocol.

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate

Several milliliters (at least 14 mL) of media were prewarmed in a 37° C., 5% CO₂ incubator (media is GWM) for ≥45 minutes prior to use.

For each isolate, two incubation tubes were prepared in PCR tubes (or two tubes in a 8-tube strip of PCR tubes), following the chart below, titled “Primary Incubation (Antibiotic Exposure)”. Add NaHCO₃ (conditioner), DNaseI (degrader). Wait to add antibiotic until bacteria cells are incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 4).

Primary Incubation (Antibiotic Exposure): (100 μL in PCR tubes) Component Volume Ng Suspension in GWM 10 μL MHB (media) 75.5 μL NaHCO₃ (100 mM) 4.5 μL (4.5 mM) 10X ABX (treated) 5 μL or NF water in control (untreated) NEB DNaseI (2000 U/mL) (degrader) 5 μL (100 U/mL)

Enhancement of accessibility after the primary incubation is done in this example with the surfactant CHAPS as the enhancer.

Secondary Incubation: DNaseI digestion in the presence of enhancer (50 μL in PCR tubes) Component Volume Ng Suspension (from primary incubation) 25 μL (50 U/mL DNaseI, 0.5X ABX, 2.5 mM NaHCO₃) CHAPS (200 mM, 20X) (enhancer) 2.5 μL (10 mM, 1X) NaHCO₃ (100 mM) 2.5 μL (2.5 mM) NEB DNaseI (degrader) 1.25 μL (50 U/mL) MHB (media) 18.75 μL

Step 1 can be performed before or after steps 2-3, or in parallel with step 4 as will be understood by a person skilled in the art upon reading of the current disclosure.

2. Providing a Sample Containing Bacteria

ng_5, ng_17, and ng_19 are ceftriaxone-susceptible isolates and ng_30 is a ceftriaxone-reduced-susceptible isolate. (see the “Clinical Isolate MICs” table for N. gonorrhoeae for MICs in the General Methods).

The bacteria were prepared according to the “NG Stock and Resuspension” description in the General Methods section with the following changes: media (GWM). incubate on shaker (1000 rpm) in 37° C., 5% CO₂ incubator (120 minutes).

While cell pellet is re-suspending, the antibiotic exposure condition (everything except Ng suspension and ABX) was prepared (see table below), and the tubes were prewarmed on a thermocycler set to 37° C. so the liquid inside the tube reaches 37° C.

3. Optionally, Pre-Process the Sample Containing Bacteria

OD600 (dilute 1/10 in media) was measured. OD of suspension was adjusted to OD=0.5 in pre-warmed MHB media+5 mM NaHCO₃

4. Add the Antibiotic

Ceftriaxone (CRO) was prepared as follows. 1.0 mg/mL ceftriaxone stock was diluted, from −80° C. storage. 20× Stock prepared (1×1 μg/mL CRO 10 μg/mL=0.020 mg/mL CRO) in a 500 μL volume by mixing 10 μL (1.000 mg/mL stock)+490 μL NF water

The antibiotic was then added to the corresponding exposure tubes setup in step 1.

5. Split the Sample into Two or More Parts

10 uL of the Ng suspension (from step 3) was withdrawn into a pipette (following the chart for the primary exposure in step 1).

6. Combine Sample with Incubation Media

The timer started and the Ng suspension was added. The time was recorded for Lab Notebook records. 10 uL of the Ng suspension was added into “treated” or “untreated” media; this addition can be performed sequentially or in parallel (using multichannel pipet).

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time. (15 Minutes; Primary Incubation)

-   -   Close primary incubation tubes, spray with 70% EtOH     -   Vortex briefly (2-4 seconds)     -   Centrifuge to remove droplets from the caps     -   Spray with 70% EtOH     -   Transport the tubes to the thermocycler (set to 37° C.)

8. Dilution of Treated and Untreated Samples into a Secondary Incubation for a Short Amount of Time (5 Minutes; Secondary Incubation)

The samples were mixed before transferring 25 μL of the sample to the prepared secondary incubation (enhancer) tubes. Follow the charts in step 1 to add the appropriate volume of cells to each tube.

-   -   Close tubes, spray with 70% EtOH     -   Vortex briefly     -   Centrifuge to remove droplets from the caps     -   Spray with 70% EtOH     -   Return to the thermocylcer for incubation at 37° C. (for 5         minutes)

9. Extraction (at 30 Minutes) of Treated and Untreated Samples to Purify DNA

Extraction was performed by following “DEB Extraction” description in the general methods using PCR tubes, and then transferring 10 μL of the contents to 90 μL of the extraction buffer.

10. Measurement of DNA by qPCR

qPCR mix was prepared as follows (see the “qPCR” protocol in the General Methods):

-   -   Primers were for the Ng 16S gene in each amplification     -   Samples were run in triplicate     -   35 cycles     -   Annealing temperature of 62° C.

Cqs measurements were used to calculate the CT ratio at each treated, untreated pair at the same conditions.

FIG. 24 compares the accessibility AST results for four isolates treated with 1 μg/mL ceftriaxone. Isolates ng_5, ng_17, and ng_19 are all susceptible to ceftriaxone, whereas, isolate ng_30 has reduced-susceptibility to ceftriaxone. The CT ratios can be used to differentiate between the antibiotic-susceptible (CT>1.5) and the antibiotic-resistant (CTR<1.5).

The results illustrate that a two-step incubation with a primary incubation of 15 min.±CRO and a secondary incubation with an enhancer of 10 mM CHAPS with DNaseI successfully differentiates between the CRO-susceptible and CRO-resistant isolates screened.

Example 12: AST Based on Nucleic Acid Accessibility, with 1-Step Incubation in Non-Lysing Conditions, Quantified Using Direct LAMP without Enhancer Present During Amplification

AST was performed based on nucleic acid accessibility, with 1-step incubation in non-lysing conditions, quantified using direct LAMP without enhancer present during amplification

In some embodiments, AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. In some embodiments, antibiotic exposure is performed in non-lysing conditions. Bacteria can be added directly to the nucleic acid amplification mix after antibiotic exposure. In some embodiments, the nucleic acid amplification mix is not fully lysing. The nucleic acid amplification mix can contain RNase or reverse transcriptase.

In this example, the AST was performed following the steps below:

1. Provide a sample containing bacteria

-   -   a. Isolates and antibiotics; in this example, AST was performed         using ec_1, ec_4, ec_38, and ec_39, antibiotic used was         ertapenem     -   b. Cell resuspension; resuspended small clump of each isolate         scraped from plate in 400 uL BHI (37° C.) in 2 mL screw cap         polypropylene tubes by pipetting     -   c. Cell growth; for each isolate added 20 uL of cell suspension         to 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes,         incubated with 500 rpm shaking at 37° C.+5% CO₂ for 4 hrs

2. Optionally, pre-process the sample containing bacteria

-   -   d. Measurement of OD; diluted all cell suspensions 10× (60 uL         into 540 uL pre-warmed BHI) in 1 cm path length polystyrene         disposable cuvettes, measured OD at 600 nm using portable         spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed         below (corrected for 10× dilution), ec_1 OD=3.3, ec_4 OD=3.4,         ec_38 OD=3.5, ec_39 OD=3.3     -   e. Cell dilution; diluted starter cultures 150× (10 uL into 1.5         mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ for 55         min

3. Provide non-lytic incubation media with and without antibiotic

-   -   f. Preparation of exposure condition; prepared 100 uL of         exposure conditions (not including cell suspension volume) with         and without antibiotic according to list below in 8-tube PCR         tube strips (USA Scientific 1402-2700); tubes were pre-warmed at         37° C.+5% CO₂ for 6 min

Component Volume MHB 70 uL ABX stock (20 ug/mL) 2.5 uL NF-H2O 7.5 uL Cell suspension 20 uL

4. Split the sample into two or more parts

-   -   g. Cell transfer; transferred 150 uL of cell suspensions to         appropriate tubes of 8-tube PCR tube strip so that transfer to         exposure condition could be performed with multi-channel pipette

5. Combine Sample with Incubation Media

-   -   h. Transfer to exposure; used multi-channel pipette to transfer         cell suspensions from 8-tube PCR tube strip into exposure         condition tube strips, exposure PCR tube strips were closed,         vortexed, quick spun, and transferred to pre-warmed thermocycler

6. Incubate the treated and untreated samples at a controlled temperature for a short amount of time

-   -   i. Exposure; exposure performed at 37° C. on BioRad CT1000         thermocycler for 15 minutes

7. Provide amplification mix, optionally containing RNase or reverse transcriptase

-   -   j. Prepared LAMP mix on ice according to the general protocol     -   k. Prepared qPCR mix on ice according to the general protocol

8. Combine incubation media containing sample with amplification mixes

-   -   l. Addition to LAMP mix; immediately after exposure, samples         were added to LAMP mix (already aliquoted into plate and on ice)         in triplicate using multichannel pipette     -   m. Addition to qPCR mix; immediately after addition to LAMP mix,         samples were added to qPCR mix (already aliquoted into plate and         on ice) in triplicate using multichannel pipette

9. Quantify nucleic acids using qPCR and qLAMP

-   -   n. LAMP was run on BioRad CFX96 instrument according to the         general protocol     -   o. qPCR was run on Roche LightCycler 96 instrument according to         the general protocol

10. Analyze data and make susceptibility call

-   -   p. Time-to-positive (TTP) for each sample is determined by         setting an amplification threshold and determining the time at         which the amplification curve of each sample crosses this         threshold. The average TTP for each sample is then determined.     -   q. The average TTPs of the treated sample is then subtracted         from the average TTP of the control sample to determine a TTP         Difference value. If this value is greater than the         pre-determined threshold for the organism/antibiotic combination         being tested, the sample is susceptible. If the value is smaller         than the pre-determined threshold then the sample is resistant.

FIGS. 25-26 represents the average control (solid lines) and average treated (dashed lines). FIG. 25 shows LAMP amplification curves for four isolates. Gray error bars represent standard deviation of triplicate samples for each time point where fluorescence was measured. The individual LAMP amplification curves for each sample are plotted in FIG. 26. LAMP can be performed without enhancer present during amplification. The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots in FIG. 25), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 27 represents the average time-to-positive for LAMP amplification for each sample as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold. In susceptible isolates (left two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible. This results in a larger difference between the average TTP of the control and treated samples than observed in samples resistant to the antibiotic (right two plots).

FIG. 28 represents the average control (solid lines) and treated (dashed lines) PCR amplification curves for four isolates. The control and treated can be used for comparison. PCR is a lytic amplification method and therefore when amplification occurs does not depend on accessibility of template NA molecules (NA=DNA+RNA).

FIG. 29 represents the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates. LAMP can be performed with RNase present during amplification to degrade RNA (as shown in this figure). The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 30 represents the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates. LAMP can be performed with reverse transcriptase (RT) present during amplification to reverse transcribe RNA into DNA, which is then amplified using LAMP (as shown in this figure). The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

The results illustrated in this example support the conclusion that AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. This is observed in FIG. 27, showing that the magnitude of the difference in TTP between the control and treated samples correctly predicts ETP susceptibility or resistance after 15 minutes of ETP exposure.

Example 13: AST Based on Nucleic Acid Accessibility, with Enhancer Present During Incubation, Quantified Using Direct LAMP without Enhancer Present During Amplification

In this example, AST is performed based on nucleic acid accessibility with enhancer present during incubation, quantified using direct LAMP without enhancer present during amplification. Antibiotic exposure can be performed in the presence of an enhancer. Bacteria can be added directly to the nucleic acid amplification mix after antibiotic exposure. The nucleic acid amplification mix can be selectively lysing.

In this example, the AST was performed following the steps below:

1. Provide a Sample Containing Bacteria

AST was performed using ec_2, ec_3, ec_40, and ec_41, antibiotic used was ertapenem. Small clump of each isolate scraped from plate was resuspended in 400 uL BHI (37° C.) in 2 mL screw cap polypropylene tubes by pipetting. For each isolate, 20 uL of cell suspension was added to 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, and incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜4 hrs

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD was measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_2 OD=3.6, ec_3 OD=3.3, ec_40 OD=3.5, ec_41 OD=3.7.

Starter cultures were diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ while preparing initial expossure conditions, secondary exposure conditions, and LAMP mix (60 min)

3. Provide non-lytic incubation media with and without antibiotic

100 uL of exposure condition (not including cell suspension volume) was prepared according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 6 min

Component Volume MHB 70 uL ABX stock (40 ug/mL) 2.5 uL NF-H2O 7.5 uL Cell suspension 20 uL

4. Optionally Provide Lytic Incubation Media

100 uL of exposure condition (not including cell suspension volume) was prepared according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 6 min

Component Volume MHB 70 uL CHAPS (100 mM) 20 uL NF-H2O 10 uL 5. Split the Sample into Two or More Parts

150 uL of cell suspensions were transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to initial exposure condition could be performed with multi-channel pipette.

6. Combine sample with non-lytic incubation media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into initial exposure condition tube strips. Initial exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler.

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Antibiotic exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 min.

8. Optionally Combine Sample with Lytic Incubation Media

20 uL of all samples was transferred to incubation media containing enhancer, samples were vortexed and quick spun.

9. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 5 min.

10. Provide amplification mix

LAMP mix was prepared on ice according to general protocol

11. Combine Incubation Media Containing Sample with Amplification Mixes

Immediately after exposure, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette.

12. Quantify nucleic acids using qLAMP

LAMP was run on BioRad CFX96 instrument according to general protocol

FIG. 31 represents the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates. Incubation can be performed with enhancer present. LAMP can be performed without enhancer present during amplification. The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 32 represents the average time-to-positive for LAMP amplification as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold. In susceptible isolates (left two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible. This results in a larger difference between the average TTP of the control and treated samples than observed in samples resistant to the antibiotic (right two plots).

The results illustrated in this example support the conclusion that AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. This is observed in FIG. 32, showing that the magnitude of the difference in TTP between the control and treated samples correctly predicts ETP susceptibility or resistance after 15 minutes of ETP exposure. FIG. 31 also shows that susceptible samples treated with antibiotic amplify earlier relative to their controls than resistant samples.

Example 14: AST Based on Nucleic Acid Accessibility, with 1-Step Incubation in Non-Lysing Conditions Followed by Filtering, Quantified Using Direct LAMP without Enhancer Present during amplification

In this Example, AST was Performed Based on Nucleic Acid Accessibility, with 1-Step incubation in non-lysing conditions followed by filtering, quantified using direct LAMP without enhancer present during amplification.

Antibiotic exposure is performed in non-lysing conditions. AST may be performed based on NA release during the incubation step. Samples can be filtered after antibiotic exposure to select for released NAs. Eluate from filtration can be added directly to the nucleic acid amplification mix after antibiotic exposure. Bulk qualitative, semi-quantitative, or quantitative NA quantification methods can be used to determine susceptibility based on stochasticity of replicate samples.

In this example, the AST was performed following the steps below:

1. Provide a Sample Containing Bacteria

AST was performed using ec_2, ec_3, ec_40, and ec_41, antibiotic used was ertapenem. Small clump of each isolate scraped from plate was resuspended in 400 uL BHI (37° C.) in 2 mL screw cap polypropylene tubes by pipetting. For each isolate, 20 uL of cell suspension was added to 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜4 hrs

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD was measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_2 OD=4.3, ec_3 OD=3.8, ec_40 OD=3.3, ec_41 OD=4.0. Starter cultures were diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ while preparing initial exposure conditions, secondary exposure conditions, and LAMP mix (60 min)

3. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure condition (not including cell suspension volume) was prepared according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO2 for 5 min

Component Volume MHB 70 uL ABX stock (40 ug/mL) 2.5 uL NF-H2O 7.5 uL Cell suspension 20 uL 4. Split the sample into two or more parts

150 uL of cell suspensions was transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to initial exposure condition could be performed with multi-channel pipette

5. Combine sample with incubation media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into initial exposure condition tube strips, initial exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler

6. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 min.

7. Filtration; Immediately after Initial Exposure Samples were Filtered Through 0.2 uM Cellulose acetate filters

40 uL of each sample was added to center of Costar Spin-X 0.22 uM cellulose acetate centrifuge filter (Sigma cat no. 8160, lot no. 08218000) using adjustable-width multichannel pipette. All samples were centrifuged at 1000 rcf for 30 sec in aerosol-tight benchtop centrifuge

8. Provide Amplification Mix

LAMP mix was prepared on ice according to general protocol

9. Combine Filter Eluate with Amplification Mix

Immediately after filtration, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette

10. Quantify Nucleic Acids Using qLAMP

LAMP was run on BioRad CFX96 instrument according to general protocol

FIG. 33 represents triplicate control (solid lines) and triplicate treated (dashed lines) LAMP amplification curves for four isolates. The control and treated curves can be used for comparison. The number of treated replicates that amplify can also be used for comparison. Because the samples are filtered, only NAs that have been released during sample incubation will be amplified. In susceptible isolates (top two plots), the sample treated with antibiotic shows more replicates amplifying because more NAs have been released during sample incubation.

As shown in this example, bulk qualitative, semi-quantitative, or quantitative NA quantification methods can be used to determine susceptibility based on stochasticity of replicate samples. This is observed in FIG. 33, showing that the number of replicate bulk LAMP samples that amplify can correctly predict ETP susceptibility or resistance after 15 minutes of exposure to ETP and filtration of samples.

Example 15: AST Based on Percentage of NAs Released During Incubation, Quantified Using Digital Nucleic Acid Amplification

In this example, AST was performed based on percentage of NAs released during incubation, quantified using digital nucleic acid amplification.

AST can be performed based on measurement DNA release during incubation with antibiotics. AST can be performed based on measurement RNA release during incubation with antibiotics. Incubation can be performed in the presence of an enhancer.

In this example, the AST was performed following the steps below:

1. Provide a Sample Containing Bacteria

AST was performed using ec_4, ec_39, antibiotic used was ertapenem. Small clump of each isolate scraped from plate was resuspended in 400 uL BHI (37° C.) in 2 mL screw cap polypropylene tubes by pipetting. For each isolate, 20 uL of cell suspension was added to 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜3 hrs

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions was diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_4 OD=2.7, ec_39 OD=2.6. Starter cultures was diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ while preparing initial exposure conditions (40 min)

3. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure condition (not including cell suspension volume) was prepared according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO2 for 6 min

Component Volume BHI 70 uL ETP (10 ug/mL) 10 uL Cell suspension 20 uL

4. Optionally Provide Lytic Incubation Media

100 uL of exposure condition (not including cell suspension volume) was prepared according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 20 min

Component Volume BHI 55 uL TNP (50 mM) 10 uL ETP (10 ug/mL) 10 uL NF-H2O 5 Sample from initial exposure 20 uL 5. Split the Sample into Two or More Parts

150 uL of cell suspensions was transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to initial exposure condition could be performed with multi-channel pipette.

6. Combine Sample with Non-Lytic Incubation Media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into initial exposure condition tube strips, initial exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler

7. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 min.

8. Optionally Combine Sample with Lytic Incubation Media

20 uL of all samples was transferred to incubation media containing enhancer, samples were vortexed and quick spun

9. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 5 min.

10. Filtration; Immediately after Initial Exposure Samples were Filtered Through 0.2 uM Cellulose Acetate Filters

40 uL of each sample was added to center of Costar Spin-X 0.22 uM cellulose acetate centrifuge filter (Sigma cat no. 8160, lot no. 08218000) using adjustable-width multichannel pipette. All samples were centrifuged at 1000 rcf for 1 minute in aerosol-tight benchtop centrifuge

11. Quantify Nucleic Acids Using Digital Nucleic Acid Quantification

DNA was quantified using ddPCR according to the general protocol. RNA was quantified using RT-ddPCR according to the general protocol.

FIG. 34 represents the percentage of DNA (top two plots) and RNA (bottom two plots) released after a 15 minute incubation with ertapenem. A second incubation can be performed with enhancer present. DNA and RNA concentration before and after filtration is determined using digital nucleic acid quantification and can be used to determine percentage of NAs released after incubation with antibiotic or after optional additional incubation with enhancer (percentage is calculated by dividing the DNA in the eluate by the total DNA or by dividing the RNA in the eluate by the total RNA). In susceptible isolates, a significantly larger percentage of NAs is released as a result of antibiotic exposure.

As shown FIG. 34 (top two plots), the percentage of DNA released (calculated relative to the total DNA concentration) correctly predicts susceptibility to ETP after 15 min of ertapenem exposure. As shown in FIG. 34 (bottom two plots), the percentage of RNA released (calculated relative to the total RNA concentration) also correctly predicts susceptibility to ETP after 15 min of ertapenem exposure.

Example 16: AST Performed with the Use of Sonication to Enhance Accessibility Differences Caused by Ertapenem Antibiotics in E. coli

This example uses sonication to increase the fraction of accessible bacterial genomes and filtration to decrease the amplification of inaccessible bacterial genomes and increase the difference between antibiotic-treated and antibiotic-untreated samples. The latter quantity is used to determine susceptibility in our AST method. The organism used in this example is the K12 MG1655 laboratory strain of Escherichia coli, which is a member of the Enterobacteriaceae and the same species as pathogenic Escherichia coli. Ertapenem is a representative carbapenem antibiotic used in current clinical practice.

In this example, the AST was performed following the steps below.

1. Obtain a log phase culture of E. coli at an optical density (OD) between 0.3 and 0.6, then dilute the culture to 5×10{circumflex over ( )}7 colony forming units (CFU) per mL. This diluted culture will be tube A.

2. Prepare four exposure solutions, “B1” through “B4”, comprising

a. 65% rich bacteria growth medium (such as Brain Heart Infusion medium) in molecular biology-grade water

b. A final concentration of 0 (for B1-B2) or 1 ug/mL (for B3-B4) of beta-lactam antibiotic. Here, ertapenem was used.

3. Spike into tube “A” a known amount of DNA molecules “L” with identical sequences and whose sequences are not found in bacterial genomes, such as commercially available lambda phage DNA. The extracellular “L” molecules serve as a control to detect errors in volumetric pipette measurements in subsequent steps. Mix tube A well.

4. Start a timer. Using a multichannel pipette, add 10 uL of tube A to each of tubes B1 through B4, noting the time of addition. Incubate tubes B1-4 at 37 Celsius.

5. After 15 minutes of incubation, sonicate tubes B2 and B4. Sonicate for 10 seconds with a peak power of 15 Watt, a duty cycle of 0.1, and a frequency of 50 cycles/burst.

6. Immediately after sonication (preferably no more than 20 minutes after the addition of A to B1-4), pass 50 uL of each of tubes B1-4 through a 0.2 micron cellulose acetate filter. This can be done by centrifuging the filters at 500 to 1000 relative centrifugal forces (rcf). Collect the filtrate for each of tubes B1-4 and call these tubes C1-C4.

7. Measure the concentrations of E. coli DNA and the spike-in DNA control in tubes B1-4 and C1-4 by performing ddPCR reactions with primers for a single-copy E. coli gene (e.g. uidA) or for the spike-in control.

8. Choose the spike-in control concentration of one of the tubes to be the standard control concentration. Then, normalize the concentration of E. coli DNA in a given tube by dividing the E. coli concentration by the amount of spike-in then multiplying by the standard control concentration.

9. Lastly, calculate the fraction of DNA that was released by antibiotic by dividing the normalized E. coli DNA concentration in the filtrate tubes (C1-4) by the normalized E. coli DNA concentrations in the corresponding feed tubes B1-4.

The above protocol was performed with Escherichia coli K12 MG1655 and a lambda DNA control from New England Biolabs. The results are shown in FIG. 35.

In this experimental design, only DNA that has been released from the cell cytoplasm upon lysis of the cell will be recovered after filtration. The strain used is susceptible to the concentration of ertapenem used. On the left column, where peak sonication power was 0 watt because sonication was not performed, the percentage of DNA released is less than the percentage released when sonication at a peak power of 15 watt occurred (right column). The percentages released are less than 90% and leave enough un-released DNA for a difference between antibiotic conditions to be measurable by the read-out method. The preceding characteristics indicate that sonication acted as an enhancing treatment. The combination of antibiotic exposure followed by sonication yielded an amount of DNA release that is greater than the sum of the release following antibiotic exposure alone and following sonication alone. This indicates that sonication increases the magnitude of the difference between an antibiotic-treated sample and a reference sample not treated with antibiotics.

Example 17: AST Performed Using Heating, Sonication or Detergent to Increase the Fraction of Bacterial Genomes Released after 15 Minutes of Beta-Lactam Action

In this example, AST was performed using heating (or pasteurization), sonication, or detergent exposure to increase the fraction of accessible bacterial genomes. This example also describes the use of filtration to decrease the amplification of inaccessible bacterial genomes and increase the difference between antibiotic-treated and antibiotic-untreated samples. The latter quantity is used to determine susceptibility in the AST method. The organism used in this example is the K12 MG1655 laboratory strain of Escherichia coli, which is a member of the Enterobacteriaceae and the same species as pathogenic Escherichia coli. Ertapenem is a representative carbapenem antibiotic used in current clinical practice.

The example was performed according to the following steps:

1. Obtain a log phase culture of E. coli at an optical density (OD) between 0.3 and 0.6, then dilute the culture to 5×10{circumflex over ( )}7 colony forming units (CFU) per mL. This diluted culture will be tube A.

2. Prepare six exposure solutions as follows:

Tube Name B1 B2 B3 B4 B5 B6 Brain Heart 65% by 65% by 65% by 65% by 65% by 65% by Infusion volume in volume in volume in volume in volume in volume in broth water water water water water water ertapenem 1.0 ug/mL 1.0 ug/mL 1.0 ug/mL Molecular Fill to 90 Fill to 90 Fill to 90 Fill to 90 Fill to 90 Fill to 90 biology uL uL uL uL uL uL grade water

3. Prepare six perturbation solutions as follows:

Tube Name C1 C2 C3 C4 C5 C6 Tris pH 7.0 50 mM 50 mM Brain Heart 65% by 65% by 65% by 65% by Infusion volume in volume in volume in volume in broth water water water water Tergitol NP 5 mM 5 mM 10 Molecular Fill to 90 Fill to 90 Fill to 90 Fill to 90 Fill to 90 Fill to 90 biology uL uL uL uL uL uL grade water

4. Start a timer. Add 10 uL of tube A to each of tubes B1 through B6, noting the time of addition. Immediately incubate tubes B1-B6 at 37 degrees Celsius.

5. After 15 minutes of incubation, move 10 uL of tubes B1-B6 to tubes C1-C6, such that B1 is transferred to C1, B2 to C2, and so forth. Vortex and spin C1-C6.

6. Perform these three series of actions simultaneously on tubes C1-C6:

7. Heat tubes C1-C2 to 80 degrees Celsius for 20 minutes. Immediately after heating, filter half of the tubes' contents through cellulose acetate filters with a 0.2 micron pore size, one filter per tube. Then move 10 uL of the filtrate from that filtering process into tubes of DNA extraction buffer, one tube per filtration. Vortex and briefly spin the extraction tubes. Heat the DNA extraction buffer tubes to 65 degrees Celsius for 6 minutes and then to 98 degrees Celsius for 4 minutes.

8. Sonicate tubes C3-C4 for 30 seconds with a peak power of 2.5 Watt, a duty cycle of 0.05, and a frequency of 50 cycles/burst. Immediately after sonication, filter half of the tubes' contents through cellulose acetate filters with a 0.2 micron pore size, one filter per tube. Then move 10 uL of the filtrate from that filtering process into tubes of DNA extraction buffer, one tube per sonication. Heat the DNA extraction buffer tubes to 65 degrees Celsius for 6 minutes and then to 98 degrees Celsius for 4 minutes.

9. Incubate tubes C5-C6 at 37 degrees Celsius for 5 minutes. Immediately after incubation, filter half of the tubes' contents through cellulose acetate filters with a 0.2 micron pore size, one filter per tube. Then move 10 uL of the filtrate from that filtering process into tubes of DNA extraction buffer, one tube per incubation. Heat the DNA extraction buffer to 65 degrees Celsius for 6 minutes and then to 98 degrees Celsius for 4 minutes.

10. Measure the concentrations of E. coli DNA and the spike-in DNA control in tubes B1-4 and C1-4 by performing ddPCR reactions with primers for a single-copy E. coli gene (e.g. uidA) or for the spike-in control.

11. Choose the spike-in control concentration of one of the tubes to be the standard control concentration. Then, normalize the concentration of E. coli DNA in a given tube by dividing the E. coli concentration by the amount of spike-in then multiplying by the standard control concentration.

12. Lastly, calculate the fraction of DNA that was released by antibiotic by dividing the normalized E. coli DNA concentration in the filtrate tubes (C1-4) by the normalized E. coli DNA concentrations in the corresponding feed tubes B1-4.

The results are shown in FIG. 36.

In this experimental design, only DNA that has been released from the cytoplasm upon lysis of the cell will be recovered after filtration. The strain used is susceptible to the concentration of ertapenem used. In all three conditions, the fraction of DNA detected in the filtrate is larger after antibiotic exposure than without antibiotic exposure. This indicates that antibiotic exposure increases the amount of accessible DNA of the target microorganism. All the percentages are less than 90% and leave enough un-released DNA for a difference between antibiotic conditions to be measurable by the read-out method. The preceding characteristics indicate that sonication, heating, and contact with TNP detergent all act as enhancing treatments.

Example 18: AST Based on Nucleic Acid Accessibility, with 1-Step Incubation in Non-Lysing Conditions, Quantified Using Direct LAMP with Enhancer Present During Amplification

In this example, AST was performed based on nucleic acid accessibility, with 1-step incubation in non-lysing conditions, quantified using direct LAMP with enhancer present during amplification

AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. Antibiotic exposure can be performed in non-lysing conditions. Bacteria can be added directly to the nucleic acid amplification mix after antibiotic exposure. The nucleic acid amplification mix can be partially lysing. The nucleic acid amplification mix contains an enhancer.

The example was performed according to the following steps.

1. Provide a Sample Containing Bacteria

AST was performed using ec_2, ec_4, ec_38 and ec_41, the antibiotic used was ertapenem. Small clump of each isolate scraped from plate was resuspended in 400 uL BHI (37° C.) in 2 mL screw cap polypropylene tubes by pipetting. For each isolate 20 uL of cell suspension was added to 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜3.5 hrs.

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_2 OD=2.4, ec_38 OD=3.2. Starter cultures were diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ for 80 min.

3. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure conditions (not including cell suspension volume) was prepared with and without antibiotic according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 5 min.

Component Volume MHB 80 uL ABX stock (20 ug/mL) 2.5 uL NF-H2O 7.5 uL Cell suspension 10 uL 4. Split the Sample into Two or More Parts

150 uL of cell suspensions were transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to exposure condition could be performed with multi-channel pipette

5. Combine Sample with Incubation Media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into exposure condition tube strips, exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler.

6. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 minutes

7. Provide Amplification Mix, Optionally Containing Enhancer

LAMP mix was prepared on ice according to the general protocol

8. Combine Incubation Media Containing Sample with Amplification Mixes.

Immediately after exposure, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette. Immediately after exposure, samples were added to LAMP mix containing enahancer (already aliquoted into plate and on ice) in triplicate using multichannel pipette

9. Quantify nucleic acids qLAMP with and without enhancer

LAMP was run on BioRad CFX96 instrument according to the general protocol

FIG. 37 represents the average control (solid lines) and treated (dashed lines) LAMP amplification curves for two isolates. LAMP can be performed with enhancer (right two plots) or without enhancer present during amplification (left two plots). The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 38 represents the average time-to-positive for LAMP amplification as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold. In susceptible isolates (left two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible. This results in a larger difference between the average TTP of the control and treated samples than observed in samples resistant to the antibiotic (right two plots). In all samples, amplification is enhanced (occurs more rapidly) with Tween present as an enhancer.

As shown in this example, AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. This is observed in FIG. 38, showing that the magnitude of the difference in TTP between the control and treated samples correctly predicts ETP susceptibility or resistance after 15 minutes of ETP exposure. In some embodiments, the nucleic acid amplification mix is not fully lysing. This is confirmed by FIG. 37 showing that susceptible samples treated with antibiotic amplify earlier relative to their controls than resistant samples. This example also shows that including Tween as an enhancer during amplification increases the speed of amplification.

Example 19: AST Based on Nucleic Acid Accessibility, with 1-Step Incubation in Non-Lysing Conditions, Quantified Using Direct LAMP with Enhancer Present During Amplification and Normalized to Lysing Extraction Condition

In this example, AST was performed based on nucleic acid accessibility, with 1-step incubation in non-lysing conditions, quantified using direct LAMP with enhancer present during amplification and normalized to lysing extraction condition.

AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. Antibiotic exposure can be performed in non-lysing conditions. Bacteria can be added directly to the nucleic acid amplification mix after antibiotic exposure. The nucleic acid amplification mix is not fully lysing. Quantification is normalized to a lysing extraction condition.

The example was performed according to the following steps.

1. Provide a Sample Containing Bacteria

AST was performed using ec_2, ec_4, ec_39, and ec_40, antibiotics used were ampicillin, ceftriaxone, and ertapenem. Small clump of each isolate scraped from plate was resuspended in 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜2 hrs.

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_2 OD=1.7, ec_4 OD=2.1, ec_39 OD=1.9, ec_40 OD=1.3. Starter cultures were diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ for 90 min.

3. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure conditions (not including cell suspension volume) were prepared with and without antibiotic according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 5 min, 20× antibiotic stock in below list was 320 ug/mL ampicillin or 40 ug/mL ceftriaxone or 10 ug/mL ertapenem

Component Volume MHB 80 uL ABX stock (20X) 5 uL NF-H2O 5 uL Cell suspension 10 uL 4. Split the Sample into Two or More Parts

150 uL of cell suspensions were transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to exposure condition could be performed with multi-channel pipette.

5. Combine Sample with Incubation Media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into exposure condition tube strips, exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler.

6. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 minutes

7. Provide Amplification Mix, Optionally Containing Enhancer

a. LAMP mix was prepared on ice according to the general protocol

8. Combine Incubation Media Containing Sample with Amplification Mixes

Immediately after exposure, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette

9. Optionally Extract Using Fully Lysing Conditions According to the General Protocol.

This step can be performed during sample incubation or after as will be understood by a skilled person upon reading of the current disclosure.

10. Quantify nucleic acids using qLAMP

LAMP was run on BioRad CFX96 instrument according to the general protocol. Samples extracted using the fully lysing condition can be quantified with samples added directly to LAMP mix or separately after

FIG. 39 represents the average control (black solid lines) and treated (black dashed lines) LAMP amplification curves for four isolates. Also shown are average control (gray solid lines) and treated (gray dashed lines) LAMP amplification curves for each sample extracted using a fully lysing condition. The control and treated curves can be used for comparison and can be normalized to the amplification curves from the fully lysed and extracted samples. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (top two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 40 represents the average time-to-positive for LAMP amplification with samples extracted using the fully lysing condition as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold. FIG. 41 shows the average time-to-positive for each sample amplified directly in LAMP mix normalized to the fully lysing condition. Normalization to this condition can be used for analysis and comparison. In susceptible isolates (ec_2 and ec_4), the sample treated with antibiotic amplifies earlier because NAs are more accessible. This results in a larger difference between the average TTP of the control and treated samples than observed in samples resistant to the antibiotic (e_39 and ec_40).

As shown in this example, AST can be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. This is observed FIG. 41, showing that the magnitude of the difference in TTP between the control and treated samples correctly predicts AMP, CRO, and ETP susceptibility or resistance after 15 minutes of ETP exposure. As seen in FIG. 40 showing that susceptible samples treated with antibiotic amplify earlier relative to their controls than resistant samples, the nucleic acid amplification mix is not fully lysing.

This example also shows that quantification is normalized to a lysing extraction condition. In particular, as seen in FIG. 41, the TTPs of samples added directly to amplification mix is normalized to the TTPs of the same samples extracted under fully lysing conditions. After normalization, susceptibility can be determined by comparing the magnitude of the difference between normalized control and treated samples.

Example 20: AST Based on Nucleic Acid Accessibility Using Spiked Urine Samples in Less than 30 Min Sample-to-Answer, with 1-Step Incubation in Non-Lysing Conditions, Quantified Using Direct LAMP with Enhancer Present During Amplification

In this example, AST is performed based on nucleic acid accessibility using spiked urine samples in less than 30 min sample-to-answer, with 1-step incubation in non-lysing conditions, quantified using direct LAMP with enhancer present during amplification.

AST may be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. Antibiotic exposure can be performed in non-lysing conditions. Bacteria can be added directly to the nucleic acid amplification mix after antibiotic exposure. The nucleic acid amplification mix can be selectively lysing. Sample matrix used in this example is urine containing bacteria. All steps from sample-to-answer are performed in less than 30 min.

The example was performed according to the following steps.

1. Provide a Sample Containing Bacteria

AST was performed using ec_4, ec_40, kp_2, and kp_8, antibiotic used was ertapenem. Small clump of each isolate scraped from plate was resuspended in 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO₂ for ˜2.2 hrs.

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, OD measured at 600 nm using portable spectrophotometer (BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_4 OD=2.4, ec_40 OD=1.8, kp_2 OD=2.7, kp_8 OD=2.8

3. Optionally, Spike Urine Sample with Bacteria

10 uL of ec_4, kp_2, and kp_8 samples were added to pre-filtered pooled human urine, 14 uL of ec_40 sample added to pre-filtered pooled human urine, incubated with 500 rpm shaking at 37° C.+5% CO₂ for 31 min

4. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure conditions (not including cell suspension volume) were prepared with and without antibiotic according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO₂ for 11 min, 20×ABX stock listed below=10 ug/mL for Ec samples and 20 ug/mL for Kp samples

Component Volume MHB 80 uL ABX stock (20X) 5 uL NF-H2O 5 uL Cell suspension in urine 10 uL 5. Split the Sample into Two or More Parts

150 uL of cell suspensions in urine were transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to exposure condition could be performed with multi-channel pipette.

6. Start Timer

Timer is started to record sample-to-answer time of all steps. This step can be performed immediately before or after combining samples with incubation media.

7. Combine Sample with Incubation Media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into exposure condition tube strips, exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler

8. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 13 minutes.

9. Provide Amplification Mix

LAMP mix was prepared on ice according to the general protocol

10. Combine Incubation Media Containing Sample with Amplification Mixes

Immediately after exposure, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette.

11. Quantify nucleic acids using qLAMP

LAMP was run on BioRad CFX96 instrument according to the general protocol

12. Analyze Once Amplification Curves have Crossed Given Threshold

Once all samples have amplified to a fluorescence value greater than set threshold (here 1000 RFU was used), amplification is stopped and time-to-positive values are determined by copying values from instrument to data analysis spreadsheet.

Susceptibility calls are returned according to magnitude of difference in TTPs between control and treated samples.

13. Stop Timer

Timer is stopped and sample-to-answer time recorded

FIG. 42 represents the average control (solid lines) and treated (dashed lines) LAMP amplification curves for four isolates. The control and treated curves can be used for comparison. LAMP is not a fully lytic amplification method and therefore when amplification occurs depends on the accessibility of template NA molecules (NA=DNA+RNA). In susceptible isolates (left two plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible.

FIG. 43 represents the average time-to-positive for LAMP amplification as determined from setting an amplification threshold and measuring the time at which the amplification curve crosses the threshold. In susceptible isolates (first and third plots), the sample treated with antibiotic amplifies earlier because NAs are more accessible. This results in a larger difference between the average TTP of the control and treated samples than observed in samples resistant to the antibiotic (second and fourth plots).

As shown in this example, AST can be performed based on nucleic acid accessibility to polymerase during the nucleic acid amplification step. This is observed in FIG. 43, showing that the magnitude of the difference in TTP between the control and treated samples correctly predicts ETP susceptibility or resistance after 15 minutes of ETP exposure. FIGS. 42-43 also demonstrate that spiked urine samples can be used as the sample type and the correct susceptibility call is still made after 15 minutes of exposure to ETP. Furthermore, the total sample-to-answer time for all steps in the above example was recorded using a timer and summed to 29.5 min.

Example 21: AST Based on Nucleic Acid Accessibility, with 1-Step Incubation and Non-Lysing Conditions Quantified with Direct LAMP, Normalized to a Fully-Lysed Sample with qLAMP Measurement

In this example, AST was performed based on nucleic acid accessibility, with 1-step incubation and non-lysing conditions quantified with direct LAMP, normalized to a fully-lysed sample with qLAMP measurement

AST may be performed with a treated sample (direct-into-qLAMP without lysis) and the DEB extracted sample (lysed sample) in qLAMP. Data may be collected on different days and data compiled. It is important to note that each DEB extracted sample and direct-to-LAMP sample must be completed in the same experiment to make the comparison for AST.

The example was performed according to the following steps.

1. Provide a Sample Containing Bacteria

AST was performed as follows:

-   -   i. kp_1, kp_2, kp_7, kp_8 (CRO, ETP, MEM)     -   ii. ec_2, ec_4, ec_39, ec_40 (AMP)     -   iii. ec 1, ec 2, ec 3, ec 4, ec 39, ec _40, ec_44, ec_45 (CRO)     -   iv. ec 1, ec 2, ec 3, ec 4, ec 38, ec 39, ec 40, ec 41, ec 44,         ec 45 (ETP)     -   v. ec_1, ec_3, ec_44, ec_45 (MEM)

Small clump of each isolate scraped from plate was resuspended in 2 mL BHI (37° C.) in 15 mL polypropylene falcon tubes, incubated with 500 rpm shaking at 37° C.+5% CO2 for ˜2 hrs

2. Optionally, Pre-Process the Sample Containing Bacteria

All cell suspensions were diluted 10× (60 uL into 540 uL pre-warmed BHI) in 1 cm path length polystyrene disposable cuvettes, measured OD at 600 nm using a portable spectrophotometer (Ultrospec 10, BioChrom cat. no. 80-2116-30), ODs listed below (corrected for 10× dilution), ec_2 OD=1.7, ec_4 OD=2.1, ec_39 OD=1.9, ec_40 OD=1.3.

Starter cultures were diluted 150× (10 uL into 1.5 mL BHI), incubated with 500 rpm shaking at 37° C.+5% CO₂ for 90 min

3. Provide Non-Lytic Incubation Media with and without Antibiotic

100 uL of exposure conditions (not including cell suspension volume) were prepared with and without antibiotic according to list below in 8-tube PCR tube strips (USA Scientific 1402-2700); tubes were pre-warmed at 37° C.+5% CO2 for 5 min, 20× antibiotic stock in below list was 320 ug/mL ampicillin or 40 ug/mL ceftriaxone or 10 ug/mL ertapenem

Component Volume MHB 80 uL ABX stock (20X) 5 uL NF-H2O 5 uL Cell suspension 10 uL 4. Split the Sample into Two or More Parts

150 uL of cell suspensions were transferred to appropriate tubes of 8-tube PCR tube strip so that transfer to exposure condition could be performed with multi-channel pipette

5. Combine Sample with Incubation Media

Multi-channel pipette was used to transfer cell suspensions from 8-tube PCR tube strip into exposure condition tube strips, exposure PCR tube strips were closed, vortexed, quick spun, and transferred to pre-warmed thermocycler.

6. Incubate the Treated Samples at a Controlled Temperature for a Short Amount of Time

Exposure was performed at 37° C. on BioRad CT1000 thermocycler for 15 minutes

7. Provide Amplification Mix, Optionally Containing Enhancer

LAMP mix was prepared on ice according to the general protocol

8. Combine Incubation Media Containing Sample with Amplification Mixes

Immediately after exposure, samples were added to LAMP mix (already aliquoted into plate and on ice) in triplicate using multichannel pipette

9. Extract Using Fully Lysing Conditions According to the General Protocol

This step can be performed during sample incubation (step 6) or after

10. Quantify Nucleic Acids Using qLAMP

LAMP was run on BioRad CFX96 instrument according to the general protocol (with tween in the amplification mix). Samples extracted using the fully lysing condition can be quantified with samples added directly to LAMP mix or separately after on a separate plate. Both TTP measurements are needed for this specific readout.

Step 9 and 10(b) can be carried out before, after, or in parallel with steps 5-7.

FIG. 44 shows the results for the AST for several E. coli and K. pneumoniae isolates with different beta lactam antibiotics. The differences between the time to positive (TTP) for the treated sample that was measured directly-into-qLAMP mix and the DEB extracted sample run in qLAMP. The DEB sample is a normalization that accounts for the total measurable nucleic acids from a fully-lysed sample and the direct LAMP measurement assesses the amount of the nucleic acids accessible to the polymerases. Differentiable AST results are obtained between the susceptible and resistant isolates when looking at the TTP Direct-TTP DEB readout. The error bars represent the error between the qLAMP triplicate wells (with standard propagation of error methods to account for the comparison of the two measurements).

As seen in this example, AST can be performed with a 1-step incubation in non-lysing conditions of a treated sample if it is compared to a fully-lysed-sample (DEB extracted) from the same initial cell suspension. Some samples are added directly to the nucleic acid amplification mix after antibiotic exposure.

Example 22: AST Performed with Calling of Susceptible Ng Samples after 15 Minutes of Antibiotic Exposure Followed by Extended Exposure to Determine Susceptibility of Samples not Called at 15 Minutes

In this example, AST was performed with calling of susceptible Ng samples after 15 minutes of antibiotic exposure followed by extended exposure to determine susceptibility of samples not called at 15 minutes.

An initial susceptibility call can be made for susceptible samples that respond after 15 min of antibiotic exposure. An extended antibiotic exposure (30 minutes total) is then used to determine susceptibility of samples, including those that do not respond to antibiotics after 15 minutes. The data in this example was obtained using the protocol described in Example 11 (two-step exposure with enhancer present during second step). In some embodiments, data may be collected on different days and data compiled.

A workflow can be designed as follows. Sample believed to contain target bacteria is incubated with antibiotic (optionally split and incubated, optionally control sample is incubated with media) for the first period of time (e.g. 15 minutes). Nucleic acid Accessibility is rapidly measured on a portion of the exposed sample, (while another portion of the sample continues to be incubated with the ABX). Optionally measuring control sample takes place. If sufficiently strong response is observed to consider the bacteria in the sample as susceptible, the test is stopped (and action is taken, for example to treat the patient). If sufficiently strong response is not observed, then after the next period of time (e.g. additional 15 minutes, or 30 minutes total ABX exposure) another portion of the treated sample is analyzed; if susceptible call is made, the test is stopped. Optionally, the cycle can continue for one or more exposure periods until the resistant, intermediate, or reduced susceptibility call can be made reliably. Rapid analysis, such as by LAMP, that can be completed in a short amount of time (e.g. within 15 minutes) is important to enable this workflow.

The same method/protocol described in Example 11 was used in this example, except for the following changes.

All samples were grown on plate for 8 hours or less. All samples were resuspended in GWM for >3 hours. Surfactant exposure time was 6.5 minutes or less. During step 8, the portion of the sample not transferred to the secondary incubation was left to incubate in the initial incubation (with antibiotic) for an additional 15 minutes (30 minutes total), after this extended incubation, a portion of this extended incubation sample was removed and handled as described in the remaining steps of the protocol (diluted into secondary incubation, etc.).

To conduct the analysis, samples were quantified using qPCR and analyzed as described in Example 11, with a percent increase in accessibility value being determined and compared to a pre-determined threshold to make the susceptibility call. The threshold used to determine susceptibility depends on the antibiotic used and the exposure time.

FIG. 45 shows compiled data for multiple samples exposed to different antibiotics for 15 minutes. Only those samples with a % DNA accessibility value above the pre-determined threshold are called susceptible. The percentage of isolates that can be called at 15 minutes depends on the antibiotic being tested.

FIG. 46 shows compiled data for multiple samples exposed to different antibiotics for 15 minutes. Only those samples with a % DNA accessibility value above the pre-determined threshold are called susceptible. The percentage of isolates that can be called at 15 minutes depends on the antibiotic being tested.

This example supports the conclusion that an initial susceptibility call can be made for susceptible samples that respond after 15 min of antibiotic exposure. This is shown in FIG. 45 where only a portion of the samples have a % DNA accessibility value large enough to be called susceptible after 15 minutes of antibiotic exposure. The example also confirms that an extended antibiotic exposure (30 minutes total) can used to determine susceptibility of samples that do not respond to antibiotics after 15 minutes. This is shown in FIG. 46, which shows that all samples can be called after 30 minutes of antibiotic exposure. The example further verifies that AST can be carried out with data collected on different days and with compiled data. The data shown in FIGS. 45-46 is a compilation of data from different days and experiments which all meets the conditions described in the methods.

Example 23: Treatment Media for Testing Procedures

In some of the experiments described herein with reference to N. gonorrhoeae, the treatment media used was Mueller Hinton Broth (MHB) from BBL Mueller Hinton II Broth Cation Adjusted (BBL cat no. 212322), which is prepared according to manufacturer instructions and autoclaved to sterilize before use, is comprised of beef extract, acid hydrolysate of casein, and starch. Mueller Hinton II Broth is cation-adjusted for calcium and magnesium ions and is used for quantitative susceptibility testing of gram-negative and gram-positive aerobic bacteria with a variety of antimicrobial agents. It is formulated to have a low thymine and thymidine content and is adjusted to the calcium and magnesium ion concentrations recommended in the CLSI standard M7-A7 (see http://legacy.bd.com/ds/technicalCenter/inserts/L007475(13).pdf, or https://www.fishersci.com/shop/products/bd-bbl-dehydrated-culture-media-mueller-hinton-ii-broth-cation-adjusted-mueller-hinton-ii-broth-cation-adjusted-500 g/b12322) at the filing date of the present disclosure)

In experiments described herein with reference to N. gonorroheae the resuspension or growing media used was HFB, MHB, or Graver-Wade Media (All concentrations based on volumes and reported concentration in Wade et al. 2007 paper) M199 cell culture medium was prepared by dissolving M199 salts (Sigma cat no. M3769, lot no. SLBW4106) in 1 L milliQ H2O. Graver-Wade salt additions to 667 mL M199 cell culture medium (final volume 1.0 L) are as follows, with the molar concentrations listed as the final concentration in the volume, 37 mM glucose, 17 mM ammonium bicarbonate, 4.9 mM Sodium acetate, 3.4 mM L-glutamine, 0.92 mM spermidine, 0.38 mM L-arginine, 0.25 mM hypoxanthine, 0.3 mM uracil, 0.25 oxaloacetate, 0.1 mM Thiamine hydrochloride, 0.04 mM L-orithine, 0.01 mM Nicotinamide adenine dinucleotide, NAD+, and ˜13 mM DL-lactate, all added to miliQ- or nuclease-free water.

In experiments described herein with reference to carbapenem-resistant Enterobacteriaceae (CREs) including E. coli and K. pneumoniae, MHB can be used for exposure media. For example, in some of the experiments described herein with reference to carbapenem-resistant Enterobacteriaceae (CREs) including E. coli and K. pneumoniae, Brain Heart Infusion (BHI) media is used for the resuspension or growth media. BHI media can be purchased as prepared, liquid media, or as dehydrated media (BD catalog number 237500). Brain Heart Infusion (BHI) is a general-purpose liquid medium used in the cultivation of fastidious and nonfastidious microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical and nonclinical materials. It serves as a base for supplemented media containing 0.1% agar, Fildes enrichment or 6.5% sodium chloride see (http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/237500.pdf) at the filing date of the present disclosure.

Example 24: Exemplary Approach for a Polymerase Accessibility Test (Pol-a-AST)

A set of experiments illustrated in Examples 25-30 was designed a rapid phenotypic AST for β-lactams based on DNA accessibility to polymerase for use with Enterobacteriaceae.

To calculate the sample size necessary to validate the method (FIGS. 50-51), the methods and Equation 5 from Banoo et al. [1] were used as described previously [2]. In particular, it was expected that the specificity and sensitivity of the pol-aAST method would be 95% with a desired margin of error of ±10%. Under these conditions, 18.2 (or 19) samples were tested with the pol-aAST method and compared to the gold standard. 36 ASTs were performed with isolates susceptible to the ABX being tested, and 46 ASTs with isolates resistant to the ABX being tested.

Example 25: Overview of Pol-aAST for Susceptible (S) and Resistant (R) Samples Exposed to β-Lactams

The pol-aAST herein described relies on differential accessibility of nucleic acids to polymerases as a result of ABX exposure. The differential accessibility to polymerase is defined as a difference in the measured rate of amplification between control and ABX-treated samples.

FIG. 47 shows an overview of an exemplary polymerase-accessibility AST (pol-aAST) for susceptible and resistant samples exposed to β-lactams.

As shown in FIG. 47, in the first step of pol-aAST, a single sample is split into control and treated aliquots of equal volume, and the treated aliquot is exposed to a β-lactam. ABX-exposure is a critical step in any phenotypic AST because phenotypic tests measure the response of bacteria to ABX. If the bacteria in the sample are resistant, it is hypothesized that no differences in NA amplification and NA quantification will be observed between control and treated aliquots. If the bacteria are susceptible, it is hypothesized that ABX treatment would lead to a compromised peptidoglycan cell wall (FIG. 47, panel a) and partial release of NAs (FIG. 47, panel b). It is hypothesized that both the compromised cell wall and partial release of NAs would increase the accessibility of NAs to polymerase in a treated ABX-susceptible aliquot. In the second step of pol-aAST, control and treated aliquots are exposed to polymerase in amplification conditions (FIG. 47, panel c) and the rate of amplification is measured.

To successfully differentiate susceptible and resistant samples, ideal amplification conditions must i) not fully lyse cells, ii) enhance alterations (damage) to the cell wall caused by exposure to β-lactams, and iii) increase NA release only from ABX-damaged cells. The rate of amplification is dependent on the concentration of polymerase-accessible NA. In susceptible samples, more NAs are released in treated aliquot, leading to faster amplification in susceptible treated aliquots (FIG. 47, panel d) relative to control. Resistant samples are not affected by the ABX, so control and treated aliquots have similar NA release and reaction time (e.g. time-to-positive). Reaction time in an isothermal amplification reaction is quantified by measuring the time-to-positive (TTP), the time it takes the reaction fluorescence to reach a predetermined threshold. It was found that using pol-aAST, isolates susceptible to the β-lactam being tested show increased accessibility of NAs to polymerase, manifesting in an earlier TTP relative to the control. The TTPs of any two samples, such as the control and treated aliquots, can be compared to generate a TTP difference value (TTPD), which can then be used to determine susceptibility by comparing to a susceptibility threshold. Here the DNA polymerase Bst 3.0 under loop-mediated isothermal amplification (LAMP) conditions was used.

Example 26: Polymerase-Accessibility AST Requires Non-Lytic Amplification Conditions

This example is carried out to test the expectation that the chemical environment in which amplification occurs would significantly impact the result of pol-aAST, and that for pol-aAST to differentiate susceptible and resistant samples, amplification conditions should not be fully lysing. To test this, pol-aAST is performed in this example using LAMP, and quantitative PCR (qPCR) (FIG. 48).

LAMP is performed at a single temperature (70° C.), which is expected not be fully lysing, whereas qPCR is a thermocycled amplification technique reaching a maximum temperature of 95° C., which is expected be fully lysing. Indeed, pol-aAST was successful in differentiating susceptible and resistant isolates when performed using LAMP, but not when performed using qPCR (FIG. 48). qPCR is tested with a total of two susceptible and two resistant isolates, none of which showed a statistically significant difference in Cq between control and treated samples. When using LAMP, detectable differences were observed between control and treated aliquots when using isolates susceptible to the target β-lactam (TTPD=1.02 min).

These differences confirm that choice of amplification chemistry is critical to the success of pol-aAST.

Example 27: Quantification of DNA Release Following Antibiotic Exposure

To investigate the mechanism of pol-aAST, experiments were performed to separate free NAs from NAs contained within structurally intact cells or associated with cell debris. Susceptible and resistant clinical isolates were exposed to β-lactams for 15 min, then filtered through 0.2 μM filters to remove cells from free NAs. NAs in the sample and eluate were then quantified using droplet digital PCR (ddPCR).

It is observed that following exposure to β-lactams, susceptible isolates treated with β-lactams released a significantly larger percentage of DNA than resistant samples (FIG. 49). The amount of DNA released depended on the ABX being tested. Exposure to meropenem resulted in an average of 21% of DNA being released from susceptible isolates, with a slightly smaller average percent (15%) released as a result of exposure to ertapenem. Interestingly, susceptible samples only released an average of 6% of DNA when exposed to CRO, demonstrating that NA release is dependent on choice of ABX and not, e.g., a universal stress response. These results also demonstrate that the magnitude of the effect of a β-lactam on cell wall integrity can be measured and is different depending on the ABX used, even on short exposure time scales.

Example 28: Validation of the Pol-aAST Using Clinical Isolates

To validate the pol-aAST method, 64 ASTs were performed using 12 clinical isolates of Escherichia coli (Ec), 8 clinical isolates of Klebsiella pneumoniae (Kp), and 5 clinical isolates of two species of Enterobacter (Ebs) and the β-lactams CRO, ETP, and MEM. The set included isolates from each genus that were susceptible and isolates that were resistant to each of the three antibiotics. In addition to isolates obtained from the UCLA Clinical Microbiology Laboratory, those tested included Ec and Kp isolates from the Centers for Disease Control (CDC) Enterobacteriaceae Carbapenem Breakpoint panel[3], as well as all available Enterobacter spp. isolates from the same panel. All samples were amplified using quantitative LAMP and categorical agreement was compared to gold-standard broth microdilution AST. Two approaches for determining susceptibility were investigated in all pol-aASTs performed.

The first approach was to compare the difference in TTP values of the control and treated aliquots in each pol-aAST as shown in FIG. 50. This difference was defined as TTPD_(CT) (FIG. 50, panel a).

Using this method, 100% categorical agreement with gold-standard AST was obtained for all antibiotics tested with Ec (FIG. 50, panel b), Kp (FIG. 50, panel c), and Ebs (FIG. 50, panel d) isolates. The values of TTPD_(CT) were well-separated between susceptible and resistant isolates in all CRE-ABX combinations. Note that the threshold values separating TTPD_(CT) of susceptible and resistant isolates depend on the antibiotic used (e.g. CRO gives a smaller response and therefore requires a lower threshold), as well as the pathogen tested (e.g. K. pneumoniae gives stronger response and requires a higher threshold). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 1.00 for all isolates and antibiotics tested. There were no errors relative to gold-standard AST when determining susceptibility by TTPD_(CT).

The second approach was to compare the difference in the TTP of a fully lysed control aliquot and the antibiotic-treated aliquot in each pol-aAST as shown in FIG. 51.

The fully lysed control aliquot was created by extracting NA from an aliquot of the ABX-treated sample using a single-step, LAMP-compatible extraction buffer. This difference was defined as TTPD_(LT) (FIG. 51, panel a). It is important to note that TTPD_(LT) only requires the antibiotic-treated sample from the exposure step, meaning the original sample does not have to be split prior to exposure. Again, the thresholds were defined individually for each antibiotic and pathogen.

To demonstrate one of the major differences between pol-aAST, a phenotypic method, and existing genotypic methods, the assay was challenged with five previously characterized isolates that had either i) no detectable β-lactamase genes or ii) lacked any genotypic signature predictive of β-lactam resistance. Two Ec and two Kp isolates were tested with no detectable β-lactamase genes as measured by both a published genotypic assay designed to screen for six β-lactamase gene families [5], as well as the Cepheid Xpert® Carba-R test (a commercial, FDA-approved genotypic assay designed to screen for five β-lactamase gene families) [8]. These four isolates did not test positive in either assay because they lack the genes these assays screen for, despite being resistant (as determined by gold-standard broth microdilution). These four tested isolates were resistant to CRO and ETP, and one isolate from each genus was also resistant to MEM. Additionally, a single resistant Ebs isolate from the CDC Enterobacteriaceae Carbapenem Breakpoint Panel (AR-Bank #0007) was tested. Whole genome sequencing of this isolate (performed by the CDC) revealed no known resistance markers [3], meaning the mechanism of resistance was uncharacterized. The pol-aAST performed excellently in all cases, and all 5 isolates were correctly categorized as resistant (Figures. 50 and 51).

Using this method, 100% categorical agreement with gold-standard AST was obtained for all antibiotics tested with Ec (FIG. 51, panel b) and Kp (FIG. 51, panel c) isolates. When testing Ebs (FIG. 51, panel d) isolates, a single error was observed in which an isolate classified as CRO-resistant was called as susceptible, resulting in an overall categorical agreement of 92%. Because of this error, the AUC for Ebs isolates tested with CRO was 0.75. Aside from this error, susceptible and resistant isolates were well separated in all cases, with AUC=1.000 for all antibiotics tested with Ec and Kp. Although a single error was observed, using the TTPD_(LT) metric still gave excellent agreement with gold-standard AST and required no splitting of the sample prior to exposure.

Example 29: Timed Sample-to-Answer Pol-aAST Using Contrived Urine Samples

To investigate the sample-to-answer time of the pol-aAST, timed experiments were performed using contrived urine samples (FIG. 52).

Sample-to-answer time is a critical metric for any assay designed to be used at the point of care (POC), but is often not reported at all, even for methods claiming to be rapid. In timed experiments, Applicant i) reduced the exposure time from 15 to 13 min to ensure all handling could be performed during the 15 min aliquoted for exposure, and ii) used an automated data-analysis spreadsheet to provide a susceptibility call as soon as the LAMP reactions reached a pre-determined threshold (indicating successful amplification). At the start of pol-aAST, a timer was started which ran for the duration of the experiment and was stopped once a susceptibility call had been made. The susceptibility of four isolates to ETP was tested simultaneously (FIG. 6a ). The pol-aAST consists of only three simple handling steps (FIG. 52, panels b-d), which allowed us to perform pol-aAST in a total time of just 29.5 min, with results in agreement with gold-standard AST (FIG. 52, panel e).

Example 30: Exemplary Testing Procedure of a Pol-aAST Performed with Clinical UTI Samples

In this example, the pol-aAST was run on clinical urine samples from patients diagnosed with UTI. These samples were confirmed to be Enterobacteriaceae-positive UTI by the UCLA Clinical Microbiology Laboratory (CML) and the pol-aASTs were run 3-5 d post-collection.

Example 28 running the pol-aAST directly on clinical urine samples reveals an insufficient response to ABX in some samples. Because those urine samples had been stored in a chemical preservative for 3-5 days after collection, some variation in the response to ABX was expected. This example was carried out to test whether the delays in the response were indeed due to the phenotypic state of bacteria in these archived samples, and not due to the intrinsic biology of the bacterial strains in these samples.

25 clinical urine specimens were obtained that exhibited an expected heterogeneity, as indicated by the wide range of urinalysis findings (see S2 Pola-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety); pH ranged from <5 to 8, specific gravities from <1.005 to >1.060 (above and below the ranges detected in standard urinalysis), protein of up to 2+, and the presence of ketones and bilirubin, as well as red blood cells, leukocytes, and squamous epithelial cells. Two of the samples were polymicrobial. To ensure a response from bacteria in these specimens, Applicant added a 30 min pre-incubation step of urine with media and increased the duration of ABX exposure to 45 min. Applicant did not optimize these conditions and did not attempt to identify the shortest possible incubation or exposure time.

Eight samples were tested for ampicillin (AMP) susceptibility and 17 samples were tested for ETP susceptibility. Prior to testing clinical samples using AMP, five E. coli isolates were tested using AMP (FIG. 54). Despite the heterogeneity in the urine matrix and the likely nutrient-deprived condition of the bacteria in the urine samples, pol-aAST experiments yielded clean separation between AMP sensitive and resistant E. coli. Additionally, Applicant was able observe a response to ETP in 14 of 17 ETP-S urine samples tested. Overall, 100% categorical agreement was obtained for determination of AMP susceptibility (4/4 susceptible, and 4/4 resistant, as shown in FIG. 53), and a response was observed indicating susceptibility to ETP in 14 of 17 (82.4%) confirmed-susceptible samples (FIG. 53), including the two polymicrobial samples. None of the samples received for testing by the pol-aAST method were ETP-resistant.

Example 31: Exemplary Approach for a Nuclease Accessibility Test (Nuc-a-AST)

A set of experiments illustrated in Examples 32-36 was performed to test a rapid phenotypic AST for β-lactams based on DNA accessibility to nuclease for use with Neisseria gonorrhoeae (Ng). These examples were carried out to test the following expectations: (i) following antibiotic exposure, DNA in susceptible cells would be more accessible to an exogenously added nuclease than DNA in resistant cells due to cell wall damage as a result of exposure to antibiotics, (ii) this difference would occur faster than cell division, and (iii) this difference in accessibility could be detected sooner if a surfactant enhancement step was included. To test the first two hypotheses, an exposure timecourse was performed using two penicillin-susceptible and two penicillin-resistant clinical isolates of Ng. To test the third hypothesis, 48 nuc-aASTs were performed using 21 clinical Ng isolates (exposed to ABX for 15 min) and 36 nuc-aASTs using 21 clinical Ng isolates (exposed to ABX for 30 min). The results were then compared to gold-standard agar dilution.

To calculate the sample size, the methods and Equation 5 from [1] were used as described previously [9]. Namely, it was suspected that the specificity and sensitivity of the nuc-aAST method would be 95% with a desired margin of error of ±10%. Under these conditions, 18.2 (or 19) samples were tested with the nuc-aAST method and compared to the gold standard. For 15 min ABX exposures, 29 ASTs were performed with isolates susceptible to the ABX being tested, and 19 ASTs with isolates resistant to the ABX being tested.

Example 32: An Exemplary Nuc-aAST Workflow Performed on a Sample Containing an Antibiotic-Susceptible Pathogen

FIG. 55 illustrates an exemplary nuc-aAST workflow for a sample containing an antibiotic-susceptible pathogen.

The nuc-aAST method measures differences in the accessibility of genomic DNA to an exogenous nuclease between control and treated samples following a short antibiotic (ABX) exposure.

Like other NA-based AST methods, the nuc-aAST (FIG. 55) relies on measuring changes in the quantity of pathogen-specific NAs in response to a treatment with ABX; however, the nuc-aAST differs from existing NA-based ASTs in three aspects.

First, in nuc-aAST, exposure of cells to β-lactams is performed in the presence of a DNase enzyme to degrade any DNase-accessible NAs (FIG. 55, panel a). DNA is accessible to DNase if it is released from the cells upon cell lysis, or if the action of the antibiotic porates the cells and allows DNase to access the intracellular DNA. Second, in nuc-aAST, an enhancement step is introduced to increase accessibility of DNA in cells that have damaged or compromised peptidoglycan caused by β-lactams; DNase is present and active during this enhancement step (FIG. 55, panel b). Third, in nuc-aAST, lysis of the sample is performed only after DNase has degraded all accessible DNA (FIG. 55, panel c). This lysis step also inactivates the DNase, so that the enzyme does not impact downstream quantification (FIG. 61). Following inactivation of DNase and lysis, DNA remaining in the sample is quantified and the percentage of accessible DNA is used to determine susceptibility (FIG. 55, panel d). The percentage of accessible DNA is quantified by subtracting the concentration of inaccessible DNA (DNA not digested) in the treated aliquot from the concentration of DNA in the control aliquot and dividing this value by the concentration of DNA in the control. Measuring the percentage of accessible DNA is an NA-based metric that enables quantification of the damage to the cellular envelope induced by ABX targeting cell wall biosynthesis.

β-lactams should primarily affect peptidoglycan [17], and should not have a major impact on the outer membrane, which serves as a structural element in Gram-negative bacteria [18]. Therefore, it is expected the primary mechanism behind any increase in accessibility to be cell lysis as a result of exposure to β-lactams, leading to release of genomic DNA to the extracellular environment containing DNase. Additionally, it is hypothesized that autolysis, which has been observed as an active stress response in Ng [19, 20], might accelerate changes in accessibility due to ABX exposure. This hypothesis was tested in a time-course experiment using two penicillin-susceptible (PEN-S) and two penicillin-resistant (PEN-R) Ng clinical isolates (FIG. 56).

A significant difference was observed in the percentage accessibility between susceptible and resistant isolates after 90 min of exposure. This is the shortest incubation time for an Ng AST with PEN to date, and faster than existing NA-based methods that rely on DNA replication [21]. However, the ideal length of an exposure step for an AST at the POC would be even shorter (15-30 min) to keep the entire workflow within the time period of a patient visit. Thus, Applicant was compelled to further accelerate changes in accessibility of DNA to nuclease as a result of β-lactam exposure in susceptible samples.

Example 33: Nuc-aAST with an Enhancer

This example was carried out to test the hypothesis that the differences in DNA accessibility observed between susceptible and resistant isolates exposed to β-lactams could be enhanced using conditions that would increase the permeability of the cell envelope.

In Gram-negative organisms like Ng, the outer membrane (OM) presents the first, and major, permeability barrier to macromolecules (e.g. nucleases and other enzymes) entering or exiting the cell, typically allowing only small molecules with molecular weights 21-600 Da to pass through [22, 23]. The peptidoglycan, in contrast to the OM, is a looser barrier that has been estimated to allow macromolecules up to 50 kDa to pass through [24-26]. Applicant thus suspected that if the OM could be compromised, damage to the peptidoglycan would result in immediate, measurable changes in accessibility of genomic DNA to DNase, both by allowing DNase to enter and by allowing DNA fragments to exit. Therefore, it is hypothesized that the OM could be compromised using an “enhancer” to decrease total assay time.

The ideal enhancer would i) increase DNA accessibility to DNase in cells that have a compromised cell wall as a result of ABX exposure, ii) result in minimal lysis of healthy cells, iii) have a consistent effect on all Ng isolates and iv) have no effect on downstream extraction and quantification of NAs. With these parameters in mind, hypo-osmotic stress, stimulated autolysis, and four classes of surfactants were selected to be tested as potential enhancers.

Hypo-osmotic stress was chosen as a method to enhance lysis of cells with damaged or compromised cell walls because osmotic stress of varying degrees is known to increase release of intracellular contents in Gram-negative bacteria, [27-29] although it has never been used to enhance accessibility in the context of AST. Cells were exposed to hypo-osmotic conditions by diluting control and treated aliquots 20-fold in water with DNase I and 500 μM CaCl₂, resulting in a ˜244 mOsm/kg shift from the ABX exposure conditions.

Autolysis was chosen as an enhancer with the rationale of leveraging an already existing stress response in Ng to enhance changes in accessibility. Autolysis is a natural stress response in Ng, and can be accelerated by incubation in high pH conditions (e.g. Tris, pH 8.5) [30, 31]. It is hypothesized that using autolysis as an enhancer might result in large changes in NA accessibility.

Surfactants were chosen as potential enhancers as a targeted chemical means of disrupting the bacterial cell membrane. A representative surfactant was selected from each of the four major charge-based classes of surfactants to investigate whether surfactant charge might lead to variability in their effectiveness due to natural variations in the OM of Ng. Applicant tested the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant benzalkonium chloride (BAC), the non-ionic surfactant TERGITOL NP (TNP), and the zwitterionic surfactant 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Each of these surfactant classes, with the exception of zwitterionic surfactants, have been well-studied for their ability to compromise the integrity of the cell envelope [32], but none have been used in the context of AST, or to change DNA accessibility on such short time scales. The less well-studied zwitterionic surfactant CHAPS was included based on the diverse interactions of zwitterionic solutes with the bacterial cell envelope [33].

Each potential enhancer was tested with respect to i) the degree of lysis caused by incubation with the enhancer alone, ii) the ability to differentiate PEN-S and PEN-R isolates using an enhancement step after exposure to PEN, and iii) the ability to differentiate CRO-S and CRO—R isolates using an enhancement step after exposure to CRO, and iv) the ability to differentiate CFM-S and CFM-R isolates using an enhancement step after exposure to CFM. PEN, CRO, and CFM were used because it it expected that the degree of change in NA accessibility as a result of enhancement would depend on the type of β-lactam used during exposure. CRO, CFM, and PEN bind and inhibit a different profile of penicillin-binding proteins [34, 35] and have different rates of killing [36], which would lead to different effects depending on the enhancer. Each enhancer was tested using multiple isolates susceptible or resistant to either PEN, CRO, or CFM. All enhancers were tested using a 5-min enhancement step after 15 min of ABX exposure. Antibiotic-exposure and enhancement steps were performed separately to decouple their effects on the Ng isolates.

Enhancers were first tested for the degree of lysis caused by a 5-min incubation with the enhancer alone (FIG. 57, panels a-f). If the enhancement step lyses the majority of cells even without antibiotic exposure, then accessibility will increase in both control and treated aliquots, and any effect of the antibiotic will be diminished. An average of <50% lysis was observed when testing all potential enhancers except BAC (FIG. 57, panel d), which showed an average of 91.6% lysis across all 12 isolates tested (See Nuca-Tables S2 and S3 of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety).

Next, the percentage accessibility was measured when using each enhancer after a 15 min exposure to PEN. The ability was evaluated to differentiate PEN-S and PEN-R isolates based on the average percentage accessibility in S isolates (which is expected to be large), the average percentage accessibility in R isolates (which is expected to be small), and the magnitude of separation between those two values. Based on these criteria, Tris (FIG. 57, panel h), TNP (FIG. 57, panel k), and CHAPS (FIG. 57, panel 1) were the most promising enhancers for differentiating PEN-S and PEN-R isolates after 15 min of exposure. However, Applicant observed differences in accessibility in response to CRO and CFM compared with PEN depending on the enhancer used (FIG. 57, panels m-x). Among CFM-S isolates, the responses to antibiotics and each enhancer were smaller than in PEN-S and CRO—S isolates. TNP and CHAPS were the only tested enhancers that enabled us to differentiate CRO-S and CRO-R responses (FIG. 57, panels w, x) after 15 min of CFM exposure. Applicant was unable to observe consistently large changes in the seven tested CRO-S isolates using the other two ionic surfactants, SDS (FIG. 57, panels i,o) and BAC (FIG. 57, panels j,p), regardless of the ABX treatment.

Following these tests, CHAPS was chosen as the enhancer to use for validation of the nuc-aAST with clinical isolates because it resulted in low percentage lysis, large increases in DNA accessibility for PEN-S and CRO—S isolates following exposure, and only small increases in the DNA accessibility of PEN-R, CRO-R, and CFM-R isolates.

Example 34: Validation of Nuc-aAST Using Clinical Isolates

To validate the nuc-aAST, 48 ASTs (with at least three biological replicates each) were performed using 21 clinical isolates of Ng exposed individually to PEN, CFM, or CRO for 15 min. The categorical susceptibility determined using the nuc-aAST was then compared to the susceptibility determined using gold-standard agar dilution (FIG. 58, panels a-c).

Receiver operating characteristic (ROC) plots [37] (FIG. 64) were created so that the area under the curve (AUC) could be calculated separately for each β-lactam tested. After 15 min of exposure we obtained an AUC of 1.000 (PEN), 0.875 (CFM), and 1.000 (CRO). The AUC is determined by scanning a threshold through the ROC plot and measuring the sensitivity and specificity at each theoretical threshold value. This scanning allows one to select the threshold that would differentiate susceptible and resistant organisms with the maximum sensitivity and specificity within the given dataset. For example, an AUC of 1.000 indicates there was a threshold value that perfectly separated susceptible (S) and resistant (R) categories. However, AUC measurements do not consider the experimental noise or the magnitude of separation between S and R samples and should be applied with care to datasets with limited numbers of measurements, such as ours. For example, in the case of CRO, the difference between the single CRO-R isolate that was available to us and the two CRO-S isolates with the lowest responses was small after 15 min of exposure. Therefore, setting the susceptibility threshold between them would be impractical, even though it would yield 100% categorical agreement. Applicant therefore decided to set a single threshold for all three ABX at a more conservative 26.5% even though this threshold generates some errors in both the CFM and CRO measurements after 15 min of ABX exposure. This threshold was chosen because it generated the fewest number of errors with 15 min CFM exposure. The exact value is the average of the closest S and R isolate.

Applicant then hypothesized that the differences observed in the magnitude of the response of the susceptible isolates after 15 min of exposure to each antibiotic, including the errors observed when testing CFM and CRO, could be the result of differences in how fast each β-lactam affects Ng [36]. For example, a possible explanation for differences among isolates in their response to ABX could be phylogenetic differences [38-40]. If isolates differ in their response times, a longer exposure would result in larger average separation between S and R isolates and potentially better categorical agreement if the S isolates were less responsive as a result of a delayed response to antibiotic.

To test the hypothesis that there are inherent differences in isolate response time, nuc-aAST was performed using CFM and CRO with 30-min exposure times and, as predicted, a larger average separation between S and R isolates and only a single error with each ABX were observed. After 15 min of exposure to CFM and CRO, 77% and 83% of susceptible isolates, respectively, were classified as susceptible using nuc-aAST. After 30 min of exposure to CFM and CRO, 95% and 92% categorical agreement was obtained for CFM and CRO, respectively. The AUC for CFM and CRO after 30 min of exposure were 0.917 and 0.981, respectively (FIG. 64).

Example 35: Pilot Nuc-aAST Tested Directly on Clinical Urine Samples

A long-term goal is to develop phenotypic AST assays and devices for clinical settings. As a proof-of-concept that the nuc-aAST method is aligned with that goal, pilot nuc-aAST experiments were performed directly on fresh clinical urine samples without a culturing step as indicated in the material and methods.

It is noted that a phenotypic Ng AST has never been successfully performed directly on clinical samples by any method; the gold standard AST requires isolation of the pathogen and then the AST is performed on the isolate. At the time Applicant began this pilot, it was unknown whether it would even be possible to obtain an AST result directly from a clinical sample without a culturing step. The stability and viability of Ng in urine samples has never been characterized, and to reduce the unknown variables, these proof-of-concept nuc-aAST were performed on fresh clinical samples. A satellite lab was set up at the AIDS Healthcare Foundation (AHF) clinic where samples were obtained.

The nuc-aAST method depends on DNase I functionality, so control experiments with a spike-in of lambda DNA into three different urine samples were first carried out to show that DNase I remained active under the conditions and time frame being tested (data shown FIG. 62). All clinical urine samples reported herein were run immediately after collection (in all four samples, the sample handling began within 30 min of sample donation). Not all clinical samples were positive or yielded AST results (some samples for negative for Ng and some could not be reliably quantified. Six nuc-aASTs were obtained directly on four clinical urine samples; including four with PEN and two with CRO. All nuc-aAST experiments were performed with technical triplicates.

To perform nuc-aAST and gold-standard comparison, each clinical urine sample was divided into two parts, one part was used immediately to run the nuc-aAST and one part was cultured to obtain the isolates for the gold-standard culture-based AST. For the nuc-aAST (FIG. 59, panels a-b), clinical urine was first centrifuged to concentrate, then resuspended in culture media with saponin (to selectively lyse host cells) and DNase I (to clear free DNA from host and dead bacterial cells) for a 15-min incubation [41-43]. The suspension was centrifuged again and then resuspended in fresh GWM media. Next, the sample was exposed to ABX (PEN or CRO) or the control solution (NF water) for 30 min. Then, all samples were exposed to the enhancer TNP for 3-5 min. Quantification was performed with qPCR and the data analyzed as described in the general protocol example. The results from each experimental replicate are shown in FIG. 59, panel d.

To perform the gold-standard culture-based AST, the second part of each of the four clinical urine samples was plated and subcultured on selective media and over the course of 3-5 days, isolates were prepared (details in “MIC testing and creation of clinical isolates” in the Methods). Gold-standard agar-dilution MIC testing was performed on these isolates and the resulting MICs are reported in Nuca-FIG. 5d and S1 Nuca-Table. It is emphasized that gold standard AST information was obtained days later after nuc-aAST experiments. The isolates from each of these four clinical urine samples were then handled identically to the urine samples, including saponin and DNase I pretreatments, and the nuc-aAST was repeated. The results of the direct-from-sample nuc-aAST and the results of the nuc-aAST on the isolate from these clinical samples are reported in FIG. 59, panel d to compare the performance. The nuc-aAST results of the clinical urine sample and the isolate carried out with the same method gave comparable results; notably, the clinical data is (as expected) more variable among replicates which is partially the result of bacterial loads in the urine sample being lower than in the assay run on isolates. A threshold at 46% accessibility (FIG. 58) correctly categorized all six ASTs from the clinical samples (S7 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety) as antibiotic-resistant or not-antibiotic-resistant.

The pilot nuc-aAST experiments with clinical samples were run with a slightly modified workflow compared with the isolates. For example, Applicant used a slightly shorter enhancement step, and TNP instead of CHAPS as the enhancer to minimize background lysis of bacterial cells. Additionally, only penicillin-intermediate (PEN-I) Ng and penicillin-resistant (PEN-R) Ng were encountered. Because one concentration of antibiotics (1 μg/mL) in the nuc-aAST was tested, which is above the MIC of the PEN-I Ng and below MIC of the PEN-R Ng, as expected PEN-I Ng gave a susceptible (or “not resistant”, NR) call and PEN-R Ng gave a resistant call.

Example 36: Sum-of-Steps Total Time Using Contrived Urine Samples

To make a more realistic measure of total assay time, the extraction and quantification steps of the nuc-aAST were modified. In this example, the exposure and enhancement steps were performed as described above, but NA quantification was performed using a rapid, chip-based, digital loop-mediated isothermal amplification (dLAMP) method, as described previously [15]. Additionally, a faster, single-step nucleic acid extraction method based on previous work [2] was used. Both modifications made the workflow faster. Additionally, the high precision of digital quantification allowed us to make a susceptibility call as soon as there was a significant difference between the concentration of NAs in the control and treated aliquots.

The total assay time was measured based on the sum of the steps of the nuc-aAST using contrived urine samples. Contrived samples mimic clinical urine samples and allowed us to better evaluate how the assay would perform in a clinical setting compared with assays performed with isolates in media. Samples were created using two PEN-S and two PEN-R isolates; one of the two PEN-R isolates was positive for β-lactamase activity, which was included in order to have PEN-R isolates with different mechanisms of resistance.

To perform the AST, samples were first split into control and treated aliquots, and incubated at 37° C. for 15 min. Next the samples were transferred to the enhancement step, and incubated for 5 min in the presence of CHAPS. Samples were then extracted as described above and dLAMP was performed in commercial chips [15]. Images were obtained in real time using a custom imaging system [44]. LAMP quantification was performed using an automated data-analysis workflow in MATLAB [15] in which images are automatically processed and positive wells counted based on a digitized mask created from the final image (FIG. 60, panel b). NA concentrations were used to determine percentage accessibility as soon as the measured NA concentrations in the susceptible sample became significantly different between the control and treated chips. All samples were tested in a total time (measured as the sum-of-steps) of 30 min and agreed with gold-standard agar dilution (FIG. 60, panel d). Performance of dLAMP was evaluated on extractions from clinical sample nuc-aAST experiments (FIG. 60, panel e). The extension of the dLAMP reaction is a result of low nucleic acid concentrations present in the clinical samples (data shown in S5 Nuca-Table of U.S. Provisional Application No. 62/968,735 incorporated herein by reference in its entirety).

Example 37 Two-Step aAST Performed with Ng Samples Wherein Both the Control Sample and the Treated Sample Include DNaseI in Both Steps and the Enhancer is a Non-Ionic Surfactant Present in the Second Step Only

The example was performed using a two-step exposure in which the first step is exposure to a beta-lactam to beta-lactam antibiotic and the second step is an addition of a non-ionic surfactant enhancer. DNaseI is present the whole time, throughout the first and second exposure.

FIG. 59 (panels a-b) describes the layout of this experiment. An NG clinical sample or clinical isolate is incubated with beta-lactam antibiotics for 30 minutes exposure to 1 μg/mL CRO or PEN followed by a 3-5 minute exposure to the non-ionic surfactant enhancer TNP.

1. Provide Incubation Media Containing Enhancer, Accessibility, with and without an Antibiotic for Each Isolate (or Clinical Sample)

For each isolate, two incubation tubes were prepared in PCR tubes (or two tubes in a 8-tube strip of PCR tube), following the chart below, titled “Primary Incubation (Antibiotic Exposure)” including DNaseI (degrader) and antibiotic until bacteria cells are incubating, so that the time from thawing the antibiotic stock is minimized before exposure (see step 4).

Primary Incubation (Antibiotic Exposure per 50 μL) Component Volume Suspension with Ng cells 41.3 μL (concentrated, saponin-treated, in fresh GWM) 2000 U/mL DNaseI 2.5 μL (100 U/mL) 10X DNaseI reaction buffer 5 μL (1X) Antibiotic (40X), NF water in 1.25 μL controls

Step 1 can be performed before or after seteps 2-3, or in parallel with step 4 as will be understood by a person skilled in the art upon reading of the current disclosure.

2. Providing a Sample Containing Bacteria

Sample #1 (clinical sample and clinical isolate) containing Ng cells are susceptible to the antibiotic ceftriaxone. Sample #4 (clinical sample and clinical isolate) containing Ng cells are resistant to the antibiotic penicillin.

The bacteria were prepared according to the “Preparation of Ng clinical sample suspensions” Either the clinical sample in urine or a clinical isolate in culture media is centrifuged, and resuspended in the saponin-treatment in fresh GWM (as detailed in the table below), incubated for 15 minutes, centrifuged, and resuspended in Fresh GWM.

Preparation of Ng clinical sample after being concentrated. (saponin-treatment, in fresh GWM) Component Volume Ng cells present in a urine 640 μL sample or resuspended in media 2000 U/mL DNaseI 40 μL (100 U/mL) 10X DNaseI reaction buffer 80 μL (1X) 20X Saponin (20%) 40 μL (1%)

Add DNaseI, 10× DNase reaction buffer to the final ratios as described in the Table in step 1.

3. Add the Antibiotic

Split the sample into two or more parts. Transfer 41.3 μL of the suspension containing Ng cells, DNaseI (degrader), and DNaseI reaction buffer into tubes warmed to 37° C. containing 1.25 μL of antibiotic (treated sample) or water (control sample) as described in the Table in step 1.

Optionally, the sample can be split into more parts to run replicate exposures of the control sample and the treated samples.

The timer started as the Ng suspension was added to the antibiotic solution. The time was recorded for Lab Notebook records. The

4. Incubate the Treated and Untreated Samples at a Controlled Temperature for a Short Amount of Time (30 Minutes at 37° C.; Primary Incubation)

-   -   Close primary incubation tubes, spray with 70% EtOH     -   Vortex briefly (2-4 seconds)     -   Centrifuge to remove droplets from the caps     -   Spray with 70% EtOH     -   Transport tubes to a heat block (set to 37° C.)

Incubate for 30 minutes at 37° C.

5. Dilution of Treated and Untreated Samples into a Secondary Incubation for a Short Amount of Time (3-5 Minutes; Secondary Incubation)

The samples were mixed before adding 2.7 μL of a non-ionic surfactant enhancer TNP to the same tube, see the Table below

-   -   Transport tubes from heat block into biological safety cabinet         (BSC)     -   Ensure all caps of tubes are closed and vortex briefly (2-4         seconds)     -   Centrifuge to remove droplets from the caps     -   Open the tubes and use a multichannel pipette to transfer 2.7 μL         of the enhancer to the control and treated tubes.     -   Close incubation tubes for the secondary incubation, spray with         70% EtOH     -   Vortex briefly (2-4 seconds)     -   Centrifuge to remove droplets from the caps     -   Spray with 70% EtOH     -   Transport tubes to a heat block (set to 37° C.)

Secondary Incubation (Antibiotic Exposure per 52.75 μL) Component Volume Suspension with Ng cells 41.3 μL (concentrated, saponin-treated, in fresh GWM) 2000 U/mL DNaseI 2.5 μL (100 U/mL) 10X DNaseI reaction buffer 5 μL (1X) Antibiotic (40X), NF water in 1.25 μL controls Enhancer 20X TNP 2.7 μL

6. Extraction (at 33-35 Minutes) of Treated and Untreated Samples to Purify DNA

Extraction was performed by following “DEB Extraction” description in the general methods using PCR tubes, by transferring 20 μL of the contents to 80 μL of the extraction buffer.

7. Measurement of DNA by qPCR

qPCR mix was prepared as follows (see the “qPCR” protocol in the General Methods):

-   -   Primers were for the Ng 16S gene in each amplification     -   Samples were run in triplicate     -   35 cycles     -   Annealing temperature of 62° C.

Cqs measurements were used to calculate the percentage DNA accessibility at each treated, untreated pair at the same conditions.

FIG. 59(d) compares the accessibility AST results for four isolates treated with 1 μg/mL ceftriaxone. Sample #1 and Sample #2 are susceptible to ceftriaxone and have penicillin-intermediate MICs whereas, Sample #3 and Sample #4 are resistant to penicillin. The mean percentage accessibility from the three replicate of each sample and sample type can be used to differentiate between the antibiotic-susceptible, and intermediate (not-resistant), samples and isolates (percentage accessibility >46%) and the antibiotic-resistant (percentage accessibility <46%).

The results illustrate that a two-step incubation with a primary incubation including DNaseI of 30 min.±CRO or PEN and a secondary incubation with an enhancer of 5 mM TNP with DNaseI successfully differentiates between the CRO-susceptible or PEN-intermediate (not-resistant) and PEN-resistant clinical samples and clinical isolates screened. DNaseI is present in both the control sample and the treated sample for the entire duration of the assay.

Example 38: Filtration AST Performed by Comparing Detected Accessible Nucleic Acid Concentration Value with an Accessible Nucleic Acid Concentration of a Reference Treated Sample and/or with a Reference Total Nucleic Acid Concentrations

This experiment was conducted to identify the in vitro dynamics of beta-lactam activity and o validate and demonstrate the AST assay's ability to determine susceptibility of the bacteria strain Escherichia coli K12.

A contrived clinical specimen was made by inoculating <1 μL of an overnight culture on LB agar of Escherichia coli K12 into Brain-Heart Infusion broth. The inoculum was small enough that no detectable difference in the sample's optical density at 600 mm (OD₆₀₀) before and after inoculation was detected by an Ultrospec 10 spectrophotometer with an analytical sensitivity of 0.01 absorbance units. After an incubation at 37° C., the batch culture became turbid with an OD₆₀₀ of 0.34 absorbance units after about 2 hours of incubation. The batch culture was diluted to 2.08 e6 CFU/mL immediately prior to contacting the cells with antibiotic. This ensured that a known density of exponential growth phase bacteria was present at the start of the AST protocol.

To begin the AST protocol, 24 μL of the above batch culture dilution was added to and mixed with 1 μL of water containing either 25 μg/mL or 0 μg/mL of dissolved ertapenem (ETP) antibiotic. The addition of bacteria to antibiotics was performed for 20 exposures in parallel by use of a multichannel pipette. 10 exposures contained ertapenem and served as treated conditions, while the other 10 exposures contained no ertapenem and served as reference conditions. The 20 antibiotic exposures were incubated at 37° C. The time elapsed between the beginning of the AST protocol and each action taken during the experiment were recorded to the nearest second by means of a stopwatch, ± up to 10 seconds for time needed to handle the stopwatch.

At approximately 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes after the beginning of the AST protocol, measured from the time of first contact with antibiotic, 14.6 μL of one treated condition antibiotic exposure and 14.6 μL one reference condition antibiotic exposure were each transferred, by multichannel pipette, to an individual sterile, polypropylene, 500 μL-capacity Corning Costar Spin-X microcentrifuge filter unit with a 0.22 μm pore size cellulose acetate filter and an accompanying collection tube. The filter units were centrifuged at 2000 rcf for 2 minutes. The fluid collected in the collection tube creates a “filtrate”.

Meanwhile, 10 μL of each antibiotic exposure was transferred to a PCR tube containing 10 μL of Lucigen™ DNA Extraction Buffer to create a “feed extraction”. The feed fractions were promptly vortexed to inhibit bacterial growth. When the centrifuging was completed, 12 μL of the filtrate was added to 12 μL of Lucigen™ DNA Extraction Buffer to create a “filtrate extraction”. Both the feed extractions and the filtrate extractions (a total of 4 per time point) were then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

The genomic DNA concentration in the 20 feed extractions and 20 filtrate extractions was measured by ddPCR. 5.0 uL of each extraction was separately added to 10 μL of BioRad™ QX200 ddPCR Evagreen supermix, 0.8 μL of a PCR primer pair at 10 μM (for a final concentration of 0.4 μM), and 4.2 μL of nuclease-free water to create a 20 μL ddPCR reaction. The primers used possessed the following sequences: 5′-GGTAGAGCACTGTTTTGGCA-3′ (SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). Droplet digital PCR was performed according to manufacturer's instructions on the BioRad QX200 ddPCR platform. Each of the 40 cycles during thermocycling comprised a 30 second denaturing step at 95° C. and a 60 second annealing and extension step at 60° C. The result of the ddPCR was 20 extracellular nucleic acid concentration values (NACV) and 20 total nucleic acid concentration values. Each set of 20 concentration values comprised 10 pairs of a treated NACV and a reference NACV. The NACVs are plotted in FIG. 65.

Reverse transcription of the feed extractions and filtrate extractions was not performed. However, the extractions were preserved at −80° C., so reverse transcription and subsequent steps of this protocol could be performed in the future if desired.

The treated extracellular NACV, the treated total NACV, the reference extracellular NACV, and the reference total NACV from the same time point can be together viewed as the result of one instance of a filtration accessibility AST with a concurrent reference condition.

Accordingly, at each time point, one can calculate the treated-to-reference ratio of the treated and reference extracellular NACVs, or another metric equivalent in function such as the relative difference.

In addition or in the alternative, one can also calculate a normalized treated value and a normalized reference value, where the extracellular NACVs are normalized by their respective total NACVs. For example, in this experiment, the normalized values take the form of the “percent extracellular”. The percent extracellular is calculated for using the formula 100*E/T, where E is the extracellular NACV and T is the total NACV (from the same time point). A graph of the percent extracellular over time is given in FIG. 66.

Accordingly, at each time point, one can also calculate a treated-to-reference ratio (or another metric equivalent in function such as the relative difference) of the normalized treated and normalized reference extracellular NACVs.

Susceptibility can be called in multiple equivalent ways. For example

-   -   a. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of “reference”/reference ratios to define a         “non-concurrent reference distribution”. Then calculate p-value         of this experiment's treated/reference ratio (of two E/I's or         two PE's) with respect to the non-concurrent reference         distribution. If p-value significant, reject null hypothesis and         call “susceptible”.     -   b. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized reference values from data to define         a “non-concurrent reference distribution”. Calculate p-value of         this experiment's normalized reference with respect to         non-concurrent reference distribution. If this experiment's         normalized reference's p-value is not significantly improbable,         proceed to calculate the p-value of this experiment's normalized         treated value. If this second p-value is significant, reject         null hypothesis and call “Susceptible”. If the first p-value (of         the normalized reference) is significantly improbable, then one         finds new prior data from experiments more similar to the         current experiment; redo the assay if the previous option is not         readily available to ensure no errors in operation; or, if the         previous option is not possible, compare the treated/reference         ratio (or metric equivalent in function such as the relative         difference) to a univariate “a priori threshold value” or APTV.         The APTV can range from 1 to infinity and reflects one's beliefs         prior to the experiment or the collection of non-concurrent         reference data, which 1 being the most generous possible         threshold that is useful, and APTVs between 1 and 8 being         preferred. If one makes the assumption that unequal random         partitioning of cells during sampling of the clinical specimen         is the only cause for any reference extracellular NACV being         higher than the treated extracellular NACV, and that one knows         the cell density of the specimen and the volumes of the treated         and reference samples, then the number of cells in each sample         will be multinomially distributed with probability parameters         equal to the proportions of the sample volumes to the total         specimen volume. Any choice of the APTV will then correspond to         a guess of the cell density and for a percentile of false         positive cases that would result given the guessed cell density.         For example, for two untreated samples X and Y, each 10 μL,         taken from a 1000 μL specimen with 4000 CFU/mL, the chance that         the number of cells in sample X is 2 or more times that of         sample Y (or vice versa) was calculated to be about 0.002754         using the statistical software R. Therefore, if an assay's         treated-reference ratio is greater than or equal to an APTV of         2.0, then there is only a 0.275% chance of a “Susceptible” call         being incorrect. If the specimen density were actually lower         than 4000 CFU/mL, and the chosen APTV remains at 2.0, then the         chance of an incorrect “Susceptible” call increases. A graph of         the false positive rate (where a “susceptible” call is         considered positive) as a function of APTV and three cell         densities is shown in FIG. 77. The choice of the APTV value is         necessarily subjective, reflects a trade-off between assay         diagnostic sensitivity and diagnostic specificity, and should be         chosen by the user to fit their specific clinical needs.     -   c. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized “reference”/reference ratios to         define a “non-concurrent reference distribution”. Then calculate         p-value of this experiment's treated/reference ratio of         normalized extracellular NACVs with respect to the         non-concurrent reference distribution. If p-value significant,         reject null hypothesis and call “susceptible”.

The susceptibility was correctly called for ASTs with exposure durations of at least 20 minutes (see FIGS. 65 and 66).

The percent of cells lysed (the percent extracellular) increased over time only when one expects there to be antibiotic killing.

One can see both the change in accessibility as well as a change in the total population of bacteria (a growth-based AST).

Example 39 Filtration AST Performed by Comparing Detected Accessible Nucleic Acid Concentration Value with an Accessible Nucleic Acid Concentration Value of a Reference Treated Sample and/or Reference Total, Nucleic Acid Detected Concentrations

This experiment was conducted to detect the effect of antibiotic concentration on the in vitro dynamics of beta-lactam activity. and to validate and demonstrate the AST assay's ability to determine the susceptibility of the bacteria strain Escherichia coli K12.

A contrived clinical specimen was made by inoculating <1 μL of an overnight culture on LB agar of Escherichia coli K12 into Brain-Heart Infusion broth. The inoculum was small enough that no detectable difference in the sample's optical density at 600 mm (OD₆₀₀) before and after inoculation was detected by an Ultrospec 10 spectrophotometer with an analytical sensitivity of 0.01 absorbance units. After an incubation at 37° C., the batch culture became turbid with an OD₆₀₀ of 0.24 absorbance units after about 2 hours of incubation. The batch culture was diluted to 2.08 e6 CFU/mL immediately prior to contacting the cells with antibiotic. This ensured that a known density of exponential growth phase bacteria was present at the start of the AST protocol.

To begin the AST protocol, 24 μL of the above batch culture dilution was added to and mixed with 1 μL of water containing either 25 μg/mL or 6.25 μg/mL of dissolved ertapenem (ETP) antibiotic. The addition of bacteria to antibiotics was performed for 20 exposures in parallel by use of a multichannel pipette. All 20 of the exposures were treated conditions, but there were two treated conditions, each with 10 antibiotic exposures. The 20 antibiotic exposures were incubated at 37° C. The time elapsed between the beginning of the AST protocol and each action taken during the experiment were recorded to the nearest second by means of a stopwatch, ± up to 10 seconds for time needed to handle the stopwatch.

At approximately 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes after the beginning of the AST protocol, measured from the time of first contact with antibiotic, 14.6 μL of one treated condition antibiotic exposure and 14.6 μL one reference condition antibiotic exposure were each transferred, by multichannel pipette, to an individual sterile, polypropylene, 500 μL-capacity Corning Costar Spin-X microcentrifuge filter unit with a 0.22 μm pore size cellulose acetate filter and an accompanying collection tube. The filter units were centrifuged at 2000 rcf for 2 minutes. The fluid collected in the collection tube creates a “filtrate”. Meanwhile, 10 μL of each antibiotic exposure was transferred to a PCR tube containing 10 μL of Lucigen® DNA Extraction Buffer to create a “feed extraction”. The feed fractions were promptly vortexed to inhibit bacterial growth. When the centrifuging was completed, 12 μL of the filtrate was added to 12 μL of Lucigen® DNA Extraction Buffer to create a “filtrate extraction”. Both the feed extractions and the filtrate extractions (a total of 4 per time point) were then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

Next, each of the 20 feed extractions and 20 filtrate extractions were separately used to generate cDNA by reverse transcription. 2.5 μL of the given extraction was mixed with 0.1 μL of 3 U/mL Lucigen® RapiDxFire thermostable reverse transcriptase, 0.5 μL of Lucigen® RapiDxFire 10× thermostable buffer, 0.25 μL of 10 mM deoxyribonucleic acid nucleotides, and 0.2 μL of a 10 μM aqueous solution of DNA primer, according to manufacturer's instructions, to create a reverse transcription reaction with a total volume of 5.0 The primer included had a sequence of 5′-TGTCTCCCGTGATAACTTTCTC-3′. The primer's sequence was complementary to the 23S ribosomal RNA in Escherichia coli and specific to the Enterobacteriaceae family. The cDNA product that would be created from this primer contained the primer sites for the future ddPCR reaction occurring later in this AST protocol. All 40 reverse transcription reactions were heated to 60° C. for 5 minutes to create cDNAs, then heated to 95° C. for 5 minutes to stop the reaction.

The genomic DNA concentrations in the 20 feed extractions and 20 filtrate extractions was measured by ddPCR. 5.0 uL of each extraction was separately added to 10 μL of BioRad® QX200 ddPCR Evagreen supermix, 0.8 μL of a PCR primer pair at 10 μM (for a final concentration of 0.4 μM), and 4.2 μL of nuclease-free water to create a 20 μL ddPCR reaction. The primers used possessed the following sequences: 5′-GGTAGAGCACTGTTTTGGCA-3′(SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′(SEQ ID NO: 12). Droplet digital PCR was performed according to manufacturer's instructions on the BioRad QX200 ddPCR platform. Each of the 40 cycles during thermocycling comprised a 30 second denaturing step at 95° C. and a 60 second annealing and extension step at 60° C. The result of the ddPCR was 20 extracellular nucleic acid concentration values (NACV) and 20 total nucleic acid concentration values. The NACVs are plotted in FIG. 67.

The 23S ribosomal RNA cDNA concentrations in the 20 feed extractions and 20 filtrate extractions were measured by ddPCR using the same protocol as for the genomic DNA in step 6, except that the template used comprised dilutions in water of the reverse transcription reactions from step 5.

For both the 40 genomic DNA and the 40 23S ribosomal RNA cDNA concentrations values, each pair of treated extracellular NACV and treated total NACV from the same time point can be together viewed as the result of one instance of a filtration accessibility AST without a concurrent reference condition.

In this case, one can substitute the reference condition data with data from similar experiments performed at a different time. It is harder to guarantee that a reference condition performed on a different clinical specimen will be similar to the reference condition performed concurrently on the same clinical specimen as the treated condition, such a substitution usually yields sufficiently accurate results so long as the same species of microorganism or close relatives are used in the non-concurrent reference condition experiments.

After gathering or creating the non-concurrent reference condition data, one may proceed to calculate the treated-to-reference ratio.

One can also calculate a normalized treated value and a normalized reference value. In this experiment, the normalized values take on the form of the “percent extracellular”. The percent extracellular is calculated for using the formula 100*E/T, where E is the extracellular NACV and T is the total NACV (from the same time point). A graph of the percent extracellular over time is given in FIG. 68.

One can also calculate a treated-to-reference ratio of the normalized treated and normalized reference extracellular NACVs.

Susceptibility can be called for example in three equivalent ways.

-   -   a. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of “reference”/reference ratios to define a         “non-concurrent reference distribution”. Then calculate p-value         of this experiment's treated/reference ratio (of two E/I's or         two PE's) with respect to the non-concurrent reference         distribution. If p-value significant, reject null hypothesis and         call “susceptible”.     -   b. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized reference values from data to define         a “non-concurrent reference distribution”. Calculate p-value of         this experiment's normalized reference with respect to         non-concurrent reference distribution. If this experiment's         normalized reference's p-value is not significantly improbable,         proceed to calculate the p-value of this experiment's normalized         treated value. If this second p-value is significant, reject         null hypothesis and call “Susceptible”. If the first p-value (of         the normalized reference) is significantly improbable, then one         finds new prior data from experiments more similar to the         current experiment; redo the assay if the previous option is not         readily available to ensure no errors in operation; or, if the         previous option is not possible, compare the treated/reference         ratio (or metric equivalent in function such as the relative         difference) to a univariate “a priori threshold value” or APTV.     -   c. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized “reference”/reference ratios to         define a “non-concurrent reference distribution”. Then calculate         p-value of this experiment's treated/reference ratio of         normalized extracellular NACVs with respect to the         non-concurrent reference distribution. If p-value significant,         reject null hypothesis and call “susceptible”.

On this basis the susceptibility is expected to be correctly called for ASTs with an exposure durations greater than 20 minutes. For an exposure duration of 20 minutes, only the 1 μg/mL susceptibility call was confident (see FIGS. 67 and 68 and related FIGS. 69 and 70).

The results illustrated in FIGS. 67 and 68 and in the related FIGS. 69 and 70 also show that less antibiotic increased the delay in appearance of lysed cells, and less antibiotic allows the total population of bacteria to reach a higher plateau.

The results illustrated in FIGS. 67 and 68 and in the related FIGS. 69 and 70 further show that the predictable shift in in vitro dynamics caused by antibiotic concentration suggests it is possible for filtration AST to quantify the rate of antibiotic killing, which conceivably could be linked to MICs or used clinically instead of MICs.

The procedures of this examples could be modified in view of the indication of the instant disclosure to obtain variations of the procedure within the scope of the disclosure. For example an additional reference sample can be provided if one substitute the reference condition data with data from similar experiments performed at a different time.

However reference data from a reference condition performed on a reference sample from a different clinical specimen will have differences compared with reference data from reference condition performed concurrently on sample from the same clinical specimen as the treated condition, which is therefore to be preferred. However even reference data from a sample from a different clinical specimen usually yields sufficiently accurate results so long as the same species of microorganism or close relatives are used in the non-concurrent reference condition experiments as will be understood ny a skilled person.

In those instances, after gathering or creating the non-concurrent reference condition data, one may proceed to calculate the treated-to-control ratio for example along the line indicated in this example.

Example 40: Filtration AST Performed on Resistant Microorganism by Comparing Detected Accessible Nucleic Acid Concentration Value with an Accessible Nucleic Acid Concentration of a Reference Treated Sample and/or with a Reference Total Nucleic Acid Concentrations

This experiment was conducted to identify in vitro dynamics of beta-lactam activity. and to validate and demonstrate our assay's ability to determine the susceptibility of a clinical isolate of carbapenem-resistant Escherichia coli.

A contrived clinical specimen was made by inoculating <1 μL of an overnight culture on LB agar of the Escherichia coli isolate into Brain-Heart Infusion broth. The inoculum was small enough that no detectable difference in the sample's optical density at 600 mm (OD₆₀₀) before and after inoculation was detected by an Ultrospec 10 spectrophotometer with an analytical sensitivity of 0.01 absorbance units. After an incubation at 37° C., the batch culture became turbid with an OD₆₀₀ of 0.16 absorbance units after about 3 hours of incubation. The batch culture was diluted to 2.08 e6 CFU/mL immediately prior to contacting the cells with antibiotic. This ensured that a known density of exponential growth phase bacteria was present at the start of the AST protocol.

To begin the AST protocol, 24 μL of the above batch culture dilution was added to and mixed with 1 μL of water containing either 25 μg/mL or 0 μg/mL of dissolved ertapenem (ETP) antibiotic. The addition of bacteria to antibiotics was performed for 20 exposures in parallel by use of a multichannel pipette. 10 exposures contained ertapenem and served as treated conditions, while the other 10 exposures contained no ertapenem and served as reference conditions. The 20 antibiotic exposures were incubated at 37° C. The time elapsed between the beginning of the AST protocol and each action taken during the experiment were recorded to the nearest second by means of a stopwatch, ± up to 10 seconds for time needed to handle the stopwatch.

At approximately 0, 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes after the beginning of the AST protocol, measured from the time of first contact with antibiotic, 14.6 μL of one treated condition antibiotic exposure and 14.6 μL one reference condition antibiotic exposure were each transferred, by multichannel pipette, to an individual sterile, polypropylene, 500 μL-capacity Corning Costar Spin-X microcentrifuge filter unit with a 0.22 μm pore size cellulose acetate filter and an accompanying collection tube. The filter units were centrifuged at 2000 rcf for 2 minutes. The fluid collected in the collection tube creates a “filtrate”.

Meanwhile, 10 μL of each antibiotic exposure was transferred to a PCR tube containing 10 μL of Lucigen® DNA Extraction Buffer to create a “feed extraction”. The feed fractions were promptly vortexed to inhibit bacterial growth. When the centrifuging was completed, 12 μL of the filtrate was added to 12 μL of Lucigen® DNA Extraction Buffer to create a “filtrate extraction”.

Both the feed extractions and the filtrate extractions (a total of 4 per time point) were then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

Next, each of the 20 feed extractions and 20 filtrate extractions were separately used to generate cDNA by reverse transcription. 2.5 μL of the given extraction was mixed with 0.1 μL of 3 U/mL Lucigen® RapiDxFire thermostable reverse transcriptase, 0.5 μL of Lucigen® RapiDxFire 10× thermostable buffer, 0.25 μL of 10 mM deoxyribonucleic acid nucleotides, and 0.2 μL of a 10 μM aqueous solution of DNA primer, according to manufacturer's instructions, to create a reverse transcription reaction with a total volume of 5.0 The primer included had a sequence of 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). The primer's sequence was complementary to the 23S ribosomal RNA in Escherichia coli and specific to the Enterobacteriaceae family. The cDNA product that would be created from this primer contained the primer sites for the future ddPCR reaction occurring later in this AST protocol. All 40 reverse transcription reactions were heated to 60° C. for 5 minutes to create cDNAs, then heated to 95° C. for 5 minutes to stop the reaction.

The genomic DNA concentration in the 20 feed extractions and 20 filtrate extractions was measured by ddPCR. 5.0 uL of each extraction was separately added to 10 μL of BioRad® QX200 ddPCR Evagreen supermix, 0.8 μL of a PCR primer pair at 10 μM (for a final concentration of 0.4 μM), and 4.2 μL of nuclease-free water to create a 20 μL ddPCR reaction. The primers used possessed the following sequences: 5′-GGTAGAGCACTGTTTTGGCA-3′ (SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). Droplet digital PCR was performed according to manufacturer's instructions on the BioRad QX200 ddPCR platform. Each of the 40 cycles during thermocycling comprised a 30 second denaturing step at 95° C. and a 60 second annealing and extension step at 60° C. The result of the ddPCR was 20 extracellular nucleic acid concentration values (NACV) and 20 total nucleic acid concentration values. Each set of 20 concentration values comprised 10 pairs of a treated NACV and a reference NACV. The NACVs are plotted in FIG. 69.

The 23S ribosomal RNA cDNA concentrations in the 20 feed extractions and 20 filtrate extractions was measured by ddPCR using the same protocol as for the genomic DNA in step 6, except that the template used comprised dilutions in water of the reverse transcription reactions from step 5.

For both the 40 genomic DNA and the 40 23S ribosomal RNA cDNA concentrations values, the treated extracellular NACV, the treated total NACV, the reference extracellular NACV, and the reference total NACV from the same time point can be together viewed as the result of one instance of a filtration accessibility AST with a concurrent reference condition.

One can calculate the treated-to-reference ratio of the treated and reference extracellular NACVs.

One can also calculate a normalized treated value and a normalized reference value. In this experiment, the normalized values take on the form of the “percent extracellular”. The percent extracellular is calculated for using the formula 100*E/T, where E is the extracellular NACV and T is the total NACV (from the same time point). A graph of the percent extracellular over time is given in FIG. 72.

One can also calculate a treated-to-reference ratio of the normalized treated and normalized reference extracellular NACVs.

Susceptibility can be called here for example in two equivalent ways.

-   -   a. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of “reference”/reference ratios to define a         “non-concurrent reference distribution”. Then calculate p-value         of this experiment's treated/reference ratio (of two E/I's or         two PE's) with respect to the non-concurrent reference         distribution. If p-value significant, reject null hypothesis and         call “susceptible”.     -   b. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized reference values from data to define         a “non-concurrent reference distribution”. Calculate p-value of         this experiment's normalized reference with respect to         non-concurrent reference distribution. If this experiment's         normalized reference's p-value is not significantly improbable,         proceed to calculate the p-value of this experiment's normalized         treated value. If this second p-value is significant, reject         null hypothesis and call “Susceptible”. If the first p-value (of         the normalized reference) is significantly improbable, then one         finds new prior data from experiments more similar to the         current experiment; redo the assay if the previous option is not         readily available to ensure no errors in operation; or, if the         previous option is not possible, compare the treated/reference         ratio (or metric equivalent in function such as the relative         difference) to a univariate “a priori threshold value” or APTV.     -   c. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized “reference”/reference ratios to         define a “non-concurrent reference distribution”. Then calculate         p-value of this experiment's treated/reference ratio of         normalized extracellular NACVs with respect to the         non-concurrent reference distribution. If p-value significant,         reject null hypothesis and call “susceptible”.

The susceptibility was correctly called as “resistant” for all of the ASTs in this experiment, although the previous example with a susceptible strain suggests that AST accuracy will increase if exposure duration is longer than 15 minutes. (See FIGS. 71 and 72)

The data reported in FIGS. 71 and 72 also show that the total population of the resistant strain continue to increase exponentially as time progressed.

The results reported in FIGS. 71 and 72 further show Both the genomic DNA (no reverse transcription) and the 23S rRNA cDNA (with reverse transcription) data the same trends, but 23S rRNA cDNA concentration values have a higher signal because the copy number of rRNA is higher than of gDNA.

The results reported in FIGS. 71 and 72 additionally show there is a small amount of background lysis equal to about 1% of the total nucleic acids. This can be confidently seen in the untreated conditions of the ASTs containing 3000 or 10000 cells. Three extracellular nucleic acid concentration values in the 23S rRNA cDNA measurements are lower than the trend. These low readings were influenced by ddPCR artifacts causing incorrect calling of positive and negative droplets or by clerical/human error.

Example 41 Validation of Filtration AST on Low Density Samples and/or Samples with Low Numbers of Susceptible Microorganism

This experiment was conducted to validate Applicant's predictions of the assay's performance on lower densities and thus lower numbers of microorganism, specifically when the microorganism is susceptible. This experiment was further conducted to validate and demonstrate the assay's ability to determine the susceptibility of the bacteria strain Escherichia coli K12.

For the purposes of demonstration, a contrived clinical specimen was made by inoculating <1 μL of an overnight culture on LB agar of Escherichia coli K12 into Brain-Heart Infusion broth. The inoculum was small enough that no detectable difference in the sample's optical density at 600 mm (OD₆₀₀) before and after inoculation was detected by an Ultrospec 10 spectrophotometer with an analytical sensitivity of 0.01 absorbance units. After an incubation at 37° C., the batch culture became turbid with an OD₆₀₀ of 0.20 absorbance units after about 2 hours of incubation. To demonstrate the performance of filtration AST as a function of number of cells analyzed, the cultures were diluted to densities of 208000, 62500, 20800, 6250, 2080, 625, 208, and 0 cells/mL immediately before the introduction of antibiotic to create 8 batch culture dilutions, the last of which also serves as a no-cell control. The densities were chosen so that an average of 10000, 5000, 1000, 500, 100, 50, 10, and 0 cells would be present in 8 samples of the specimen.

To begin the AST protocol, 24 μL of the above batch culture dilution was added to and mixed with 1 μL of water containing either 25 μg/mL or 0 μg/mL of dissolved ertapenem (ETP) antibiotic. The addition of bacteria to antibiotics was performed for 16 exposures in parallel by use of a multichannel pipette. 8 exposures contained ertapenem and served as treated conditions, while the other 8 exposures contained no ertapenem and served as reference conditions. Each series of 8 contained one of the 8 batch culture dilutions each. The 16 antibiotic exposures were incubated at 37° C. The time elapsed between the beginning of the AST protocol and each action taken during the experiment were recorded to the nearest second by means of a stopwatch, ± up to 10 seconds for time needed to handle the stopwatch.

At approximately 30 minutes after the beginning of the AST protocol, measured from the time of first contact with antibiotic, 14.6 μL of all antibiotic exposures were separately transferred, by multichannel pipette, to individual sterile, polypropylene, 500 μL-capacity Corning Costar Spin-X microcentrifuge filter units with a 0.22 μm pore size cellulose acetate filter and an accompanying collection tube. The filter units were centrifuged at 4000 rcf for 2 minutes. The fluid collected in the collection tube creates a “filtrate”. Meanwhile, 10 μL of each antibiotic exposure was transferred to a PCR tube containing 10 μL of Lucigen® DNA Extraction Buffer to create a “feed extraction”. The feed fractions were promptly vortexed to inhibit bacterial growth. When the centrifuging was completed, 12 μL of the filtrate was added to 12 μL of Lucigen® DNA Extraction Buffer to create a “filtrate extraction”. Both the feed extractions and the filtrate extractions (a total of 4 per time point) were then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

Next, each of the 16 feed extractions and 16 filtrate extractions were separately used to generate cDNA by reverse transcription. 2.5 μL of the given extraction was mixed with 0.1 μL of 3 U/mL Lucigen® RapiDxFire thermostable reverse transcriptase, 0.5 μL of Lucigen® RapiDxFire 10× thermostable buffer, 0.25 μL of 10 mM deoxyribonucleic acid nucleotides, and 0.2 μL of a 10 μM aqueous solution of DNA primer, according to manufacturer's instructions, to create a reverse transcription reaction with a total volume of 5.0

The primer included had a sequence of 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). The primer's sequence was complementary to the 23S ribosomal RNA in Escherichia coli and specific to the Enterobacteriaceae family. The cDNA product that would be created from this primer contained the primer sites for the future ddPCR reaction occurring later in this AST protocol. All 32 reverse transcription reactions were heated to 60° C. for 5 minutes to create cDNAs, then heated to 95° C. for 5 minutes to stop the reaction.

The genomic DNA concentration in the 16 feed extractions and 16 filtrate extractions was measured by ddPCR. 5.0 uL of each extraction was separately added to 10 μL of BioRad® QX200 ddPCR Evagreen supermix, 0.8 μL of a PCR primer pair at 10 μM (for a final concentration of 0.4 μM), and 4.2 μL of nuclease-free water to create a 20 μL ddPCR reaction. The primers used possessed the following sequences: 5′-GGTAGAGCACTGTTTTGGCA-3′ (SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′(SEQ ID NO: 12). Droplet digital PCR was performed according to manufacturer's instructions on the BioRad QX200 ddPCR platform. Each of the 40 cycles during thermocycling comprised a 30 second denaturing step at 95° C. and a 60 second annealing and extension step at 60° C. The result of the ddPCR was 16 extracellular nucleic acid concentration values (NACV) and 16 total nucleic acid concentration values, each set of 16 concentration values comprising 8 pairs of a treated NACV and a corresponding reference NACV. The NACVs are plotted in FIG. 73.

The 23S ribosomal RNA cDNA concentrations in the 20 feed extractions and 20 filtrate extractions were measured by ddPCR using the same protocol as for the genomic DNA in step 6, except that the template used comprised dilutions in water of the reverse transcription reactions from step 4.

For both the thirty-two genomic DNA and the thirty-two 23S ribosomal RNA cDNA concentrations values, the treated extracellular NACV, the treated total NACV, the reference extracellular NACV, and the reference total NACV from the same batch culture dilution can be together viewed as the result of one instance of a filtration accessibility AST with a concurrent reference condition.

One can calculate the treated-to-reference ratio of the treated and reference extracellular NACVs.

One can also calculate a normalized treated value and a normalized reference value. In this experiment, the normalized values take on the form of the “percent extracellular”. The percent extracellular is calculated for using the formula 100*E/T, where E is the extracellular NACV and T is the total NACV (from the same time point). A graph of the percent extracellular over time is given in FIG. 74.

One can also calculate a treated-to-reference ratio of the normalized treated and normalized reference extracellular NACVs.

Susceptibility can be called for example in two equivalent ways.

-   -   a. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of “reference”/reference ratios to define a         “non-concurrent reference distribution”. Then calculate p-value         of this experiment's treated/reference ratio (of two E/I's or         two PE's) with respect to the non-concurrent reference         distribution. If p-value significant, reject null hypothesis and         call “susceptible”.     -   b. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized reference values from data to define         a “non-concurrent reference distribution”. Calculate p-value of         this experiment's normalized reference with respect to         non-concurrent reference distribution. If this experiment's         normalized reference's p-value is not significantly improbable,         proceed to calculate the p-value of this experiment's normalized         treated value. If this second p-value is significant, reject         null hypothesis and call “Susceptible”. If the first p-value (of         the normalized reference) is significantly improbable, then one         finds new prior data from experiments more similar to the         current experiment; redo the assay if the previous option is not         readily available to ensure no errors in operation; or, if the         previous option is not possible, compare the treated/reference         ratio (or metric equivalent in function such as the relative         difference) to a univariate “a priori threshold value” or APTV.     -   c. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized “reference”/reference ratios to         define a “non-concurrent reference distribution”. Then calculate         p-value of this experiment's treated/reference ratio of         normalized extracellular NACVs with respect to the         non-concurrent reference distribution. If p-value significant,         reject null hypothesis and call “susceptible”.

Susceptibility was correctly called for each of the 8 batch culture dilutions, except for the AST containing 10 cells and the AST containing 30 cells when RT was not used.

This suggests that using RT and rRNA as a high copy target allows one can reliably perform this assay from 10 cells and possibly fewer. Without RT, the assay is inaccurate between 30-100 cells.

A similar percentage of lysed cells is seen in the susceptible treated condition regardless of the initial density of cells. This means we do not have to worry about the inoculum effect for described in the literature.

There is a small amount of background lysis equal to between 0.2% and 0.7% of the total nucleic acids. This can be confidently seen in the untreated conditions of the ASTs containing 3000 or 10000 cells.

Example 42 Validation of Filtration AST on Low Density Samples and/or Samples with Low Numbers of Resistant Microorganism

This experiment was conducted to validate the predictions of the assay's performance on lower densities and lower numbers of microorganism, specifically when the microorganism is resistant. This experiment was also conducted to validate and demonstrate the assay's ability to determine the susceptibility of a clinical isolate of carbapenem-resistant Escherichia coli.

For the purposes of demonstration, a contrived clinical specimen was made by inoculating <1 μL of an overnight culture on LB agar of Escherichia coli K12 into Brain-Heart Infusion broth. The inoculum was small enough that no detectable difference in the sample's optical density at 600 mm (OD₆₀₀) before and after inoculation was detected by an Ultrospec 10 spectrophotometer with an analytical sensitivity of 0.01 absorbance units. After an incubation at 37° C., the batch culture became turbid with an OD600 of 0.

27 absorbance units after about 2 hours of incubation. To demonstrate the performance of filtration AST as a function of number of cells analyzed, the cultures were diluted to densities of 208000, 62500, 20800, 6250, 2080, 625, 208, and 0 cells/mL immediately before the introduction of antibiotic to create 8 batch culture dilutions, the last of which also serves as a no-cell control. The densities were chosen so that an average of 10000, 5000, 1000, 500, 100, 50, 10, and 0 cells would be present in 8 samples of the specimen.

To begin the AST protocol, 24 μL of the above batch culture dilution was added to and mixed with 1 μL of water containing either 25 μg/mL or 0 μg/mL of dissolved ertapenem (ETP) antibiotic. The addition of bacteria to antibiotics was performed for 16 exposures in parallel by use of a multichannel pipette. 8 exposures contained ertapenem and served as treated conditions, while the other 8 exposures contained no ertapenem and served as reference conditions. Each series of 8 contained one of the 8 batch culture dilutions each. The 16 antibiotic exposures were incubated at 37° C. The time elapsed between the beginning of the AST protocol and each action taken during the experiment were recorded to the nearest second by means of a stopwatch, ± up to 10 seconds for time needed to handle the stopwatch.

At approximately 30 minutes after the beginning of the AST protocol, measured from the time of first contact with antibiotic, 14.6 μL of all antibiotic exposures were separately transferred, by multichannel pipette, to individual sterile, polypropylene, 500 μL-capacity Corning Costar Spin-X microcentrifuge filter units with a 0.22 μm pore size cellulose acetate filter and an accompanying collection tube. The filter units were centrifuged at 4000 rcf for 2 minutes. The fluid collected in the collection tube creates a “filtrate”. Meanwhile, 10 μL of each antibiotic exposure was transferred to a PCR tube containing 10 μL of Lucigen® DNA Extraction Buffer to create a “feed extraction”. The feed fractions were promptly vortexed to inhibit bacterial growth. When the centrifuging was completed, 12 μL of the filtrate was added to 12 μL of Lucigen® DNA Extraction Buffer to create a “filtrate extraction”. Both the feed extractions and the filtrate extractions (a total of 4 per time point) were then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

Next, each of the 16 feed extractions and 16 filtrate extractions were separately used to generate cDNA by reverse transcription. 2.5 μL of the given extraction was mixed with 0.1 μL of 3 U/mL Lucigen® RapiDxFire thermostable reverse transcriptase, 0.5 μL of Lucigen® RapiDxFire 10× thermostable buffer, 0.25 μL of 10 mM deoxyribonucleic acid nucleotides, and 0.2 μL of a 10 μM aqueous solution of DNA primer, according to manufacturer's instructions, to create a reverse transcription reaction with a total volume of 5.0 The primer included had a sequence of 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). The primer's sequence was complementary to the 23S ribosomal RNA in Escherichia coli and specific to the Enterobacteriaceae family. The cDNA product that would be created from this primer contained the primer sites for the future ddPCR reaction occurring later in this AST protocol. All 32 reverse transcription reactions were heated to 60° C. for 5 minutes to create cDNAs, then heated to 95° C. for 5 minutes to stop the reaction.

The genomic DNA concentration in the 16 feed extractions and 16 filtrate extractions was measured by ddPCR. 5.0 uL of each extraction was separately added to 10 μL of BioRad® QX200 ddPCR Evagreen supermix, 0.8 μL of a PCR primer pair at 10 μM (for a final concentration of 0.4 μM), and 4.2 μL of nuclease-free water to create a 20 μL ddPCR reaction. The primers used possessed the following sequences: 5′-GGTAGAGCACTGTTTTGGCA-3′ (SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′(SEQ ID NO: 12). Droplet digital PCR was performed according to manufacturer's instructions on the BioRad QX200 ddPCR platform. Each of the 40 cycles during thermocycling comprised a 30 second denaturing step at 95° C. and a 60 second annealing and extension step at 60° C. The result of the ddPCR was 16 extracellular nucleic acid concentration values (NACV) and 16 total nucleic acid concentration values, each set of 16 concentration values comprising 8 pairs of a treated NACV and a corresponding reference NACV. The NACVs are plotted in FIG. 75.

The 23S ribosomal RNA cDNA concentrations in the 20 feed extractions and 20 filtrate extractions were measured by ddPCR using the same protocol as for the genomic DNA in step 6, except that the template used comprised dilutions in water of the reverse transcription reactions from step 4.

For both the thirty-two genomic DNA and the thirty-two 23S ribosomal RNA cDNA concentrations values, the treated extracellular NACV, the treated total NACV, the reference extracellular NACV, and the reference total NACV from the same batch culture dilution can be together viewed as the result of one instance of a filtration accessibility AST with a concurrent reference condition.

One can calculate the treated-to-reference ratio of the treated and reference extracellular NACVs.

One can also calculate a normalized treated value and a normalized reference value. In this experiment, the normalized values take on the form of the “percent extracellular”. The percent extracellular is calculated for using the formula 100*E/T, where E is the extracellular NACV and T is the total NACV (from the same time point). A graph of the percent extracellular over time is given in FIG. 76.

One can also calculate a treated-to-reference ratio of the normalized treated and normalized reference extracellular NACVs.

Susceptibility can be called for example in the following equivalent ways.

-   -   a. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of “reference”/reference ratios to define a         “non-concurrent reference distribution”. Then calculate p-value         of this experiment's treated/reference ratio (of two E/I's or         two PE's) with respect to the non-concurrent reference         distribution. If p-value significant, reject null hypothesis and         call “susceptible”.     -   b. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized reference values from data to define         a “non-concurrent reference distribution”. Calculate p-value of         this experiment's normalized reference with respect to         non-concurrent reference distribution. If this experiment's         normalized reference's p-value is not significantly improbable,         proceed to calculate the p-value of this experiment's normalized         treated value. If this second p-value is significant, reject         null hypothesis and call “Susceptible”. If the first p-value (of         the normalized reference) is significantly improbable, then one         finds new prior data from experiments more similar to the         current experiment; redo the assay if the previous option is not         readily available to ensure no errors in operation; or, if the         previous option is not possible, compare the treated/reference         ratio (or metric equivalent in function such as the relative         difference) to a univariate “a priori threshold value” or APTV.     -   c. Gather/create reference data from non-concurrent but similar         experiments, preferably with similar taxa. Calculate         distribution of normalized “reference”/reference ratios to         define a “non-concurrent reference distribution”. Then calculate         p-value of this experiment's treated/reference ratio of         normalized extracellular NACVs with respect to the         non-concurrent reference distribution. If p-value significant,         reject null hypothesis and call “susceptible”.

Without reverse transcription, susceptibility was correctly called for ASTs at about 19 cells and above based on the results reported in FIGS. 75 and 76. With reverse transcription, susceptibility was correctly called for ASTs at about 6 cells and above based on the results reported in FIGS. 75 and 76.

Example 43: High Throughput Antibiotic AST

High throughput AST on multiple antibiotics can be performed by simultaneously running the method of any one of the examples 1 through 42 in parallel using high-throughput instrumentation. An exemplary procedure is provided below.

Before the start of the antibiotic exposure, a Millipore MultiScreenHTS™ 96-well sterile polystyrene filter plate (Millipore-Sigma MSGVS2210) was prepared. Each well of the filter plate contained a sterile hydrophilic polyvinylidene fluoride (PVDF) filter membrane with a 0.2 μm pore size. The filter membranes were prepared by washing 50 μL of 50 μg/mL salmon sperm DNA blocking solution through the filters via a vacuum manifold. Next, any residual wash liquid was evaporated before the start of the experiment by drying the plate at 37° C. (any temperature between 4° C. and 65° C. is suitable) in a sterile environment until the membranes were visibly dry.

For the purposes of demonstration, a clinical specimen is created by resuspending a colony, grown overnight on Mueller-Hinton agar, of an Escherichia coli clinical isolate “strain X”, with an unknown susceptibility profile, in Brain Heart Infusion broth to create a batch culture. The batch culture is incubated at 35° C. for at least 45 min or until visibly turbid to allow the bacteria to enter an exponential growth phase. Then, the batch culture is diluted with Brain Heart Infusion broth to a density matching a 0.5 McFarland standard. A 0.5 McFarland standard has an absorbance at 625 nm, with a 1 cm light path, that lies between 0.08 to 0.13. Within 0-30 minutes of standardizing the batch culture density, the standardized batch culture is then diluted 100-fold, yielding an approximate density of 1000 cells/μL.

A 96 well microtiter plate was prepared with growth media and differing amounts of several beta-lactam antibiotics. Each well contained 50 μL of cation-adjusted Mueller-Hinton broth (CA-MHB) growth media and antibiotics at the twice the final concentration shown in the diagram, so that the final concentration of antibiotic after the addition of another 50 μL of batch culture would be the value shown in the diagram. Eighteen distinct beta-lactam antibiotics and beta-lactam/beta-lactamase inhibitor combinations are represented, namely ampicillin (AMP), ampicillin-sulbactam (SAM), aztreonam (ATM), cefazolin (CFZ), cefepime (FEP), cefotaxime (CTX), ceftazidime (CAZ), ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), ceftriaxone (CRO), doripenem (DOR), ertapenem (ETP), imipenem (IPM), meropenem (MEM), meropenem-vaborbactam (MEV), piperacillin (PIP), piperacillin-tazobactam (TZP), and ticarcillin-clavulanic acid (TIM). Each antibiotic is tested over a range of a 2-fold dilution series of concentrations, ranging from 2 to 8 distinct concentrations. There are 8 no-antibiotic control conditions. The CLSI breakpoint concentrations for each antibiotic are represented for each of the antibiotics

a. The choice of antibiotics and antibiotic concentrations can be adapted for different clinical needs. The arrangement of the wells can also be altered without changing the outcome of the protocol.

Col 1 Col 2 Col 3 Col 4 Col 5 Col 6 Row A AMP, ATM, FEP, CTX, CZA, CRO, 8 μg/mL 8 μg/mL 4 μg/mL 16 μg/mL 4 μg/mL 1 μg/mL Row B AMP, ATM, FEP, CTX, CZA, CRO, 16 μg/mL 16 μg/mL 8 μg/mL 32 μg/mL 8 μg/mL 2 μg/mL Row C SAM, CFZ, FEP, CAZ, CZA, CRO, 4/2 μg/mL 1 μg/mL 16 μg/mL 1 μg/mL 16 μg/mL 4 μg/mL Row D SAM, CFZ, FEP, CAZ, C/T, CRO, 8/4 μg/mL 2 μg/mL 32 μg/mL 2 μg/mL 2/4 μg/mL 8 μg/mL Row E SAM, CFZ, CTX, CAZ, C/T, CRO, 16/8 μg/mL 4 μg/mL 1 μg/mL 4 μg/mL 4/4 μg/mL 16 μg/mL Row F ATM, CFZ, CTX, CAZ, C/T, CRO, 1 μg/mL 8 μg/mL 2 μg/mL 8 μg/mL 8/4 μg/mL 32 μg/mL Row G ATM, CFZ, CTX, CAZ, C/T, DOR, 2 μg/mL 16 μg/mL 4 μg/mL 16 μg/mL 16/4 μg/mL 0.25 μg/mL Row H ATM, FEP, CTX, CZA, CRO, DOR, 4 μg/mL 2 μg/mL 8 μg/mL 2 μg/mL 0.5 μg/mL 0.5 μg/mL Col 7 Col 8 Col 9 Col 10 Col 11 Col 12 Row A DOR, ETP, MEM, TZP, MEV, none, 1 μg/mL 8 μg/mL 2 μg/mL 32 μg/mL 0.016/8 μg/mL 0 μg/mL Row B DOR, IPM, MEM, TZP, MEV, none, 2 μg/mL 0.5 μg/mL 4 μg/mL 64 μg/mL 0.063/8 μg/mL 0 μg/mL Row C DOR, IPM, MEM, TZP, MEV, none, 4 μg/mL 1 μg/mL 8 μg/mL 128 μg/mL 0.25/8 μg/mL 0 μg/mL Row D ETP, IPM, PIP, TIM, MEV, none, 0.25 μg/mL 2 μg/mL 16 μg/mL 8 μg/mL 1/8 μg/mL 0 μg/mL Row E ETP, IPM, PIP, TIM, MEV, none, 0.5 μg/mL 4 μg/mL 32 μg/mL 16 μg/mL 2/8 μg/mL 0 μg/mL Row F ETP, IPM, PIP, TIM, MEV, none, 1 μg/mL 8 μg/mL 64 μg/mL 32 μg/mL 4/8 μg/mL 0 μg/mL Row G ETP, MEM, TZP, TIM, MEV, none, 2 μg/mL 0.5 μg/mL 8 μg/mL 64 μg/mL 8/8 μg/mL 0 μg/mL Row H ETP, MEM, TZP, TIM, MEV, none, 4 μg/mL 1 μg/mL 16 μg/mL 128 μg/mL 16/8 μg/mL 0 μg/mL

To begin the AST protocol, 50 μL of the above bacteria batch culture is separately added to and mixed with each well's contents to create 96 antibiotic exposures (with the no-antibiotic controls included in the tally) each with a volume of 100 The antibiotic exposures are then incubated at 37° C. for 60 minutes while shaking at 700 rpm.

After the 60-minute incubation, 70 μL of each antibiotic exposure is separately transferred (in parallel via a multichannel pipettor or an automated pipetting station) to the 96-well filter plate. A vacuum manifold unit is used to move the 70 μL of fluid through the filter membrane and into a clean 96-well collecting plate. The collected fluid is called the “filtrate” While the filtration is ongoing, 25 μL of each the remaining antibiotic exposures (now called the “feed”) is transferred (in parallel via a multichannel pipettor or an automated pipetting station) to another 96-well plate already containing 50 μL of Lucigen™ DNA Extraction Buffer in each well, creating 96 “feed extractions”. The feed fractions are promptly vortexed to inhibit further nucleic acid synthesis by the bacteria. When the vacuum filtration is complete, 60 μL of the collected filtrate is added to and mixed with 60 μL of Lucigen™ DNA Extraction Buffer to create a “filtrate extraction”. The 96 pairs of feed extractions and the filtrate extractions are then incubated at 65° C. for 6 minutes and then at 98° C. for 4 minutes.

a. The filter pore size was chosen to prevent the passage of intact bacterial cells, which are all larger than 0.2 with rare exceptions. The centrifugation speed was chosen to be low enough to prevent cell lysis. It is expected that the filtrate will contain all or most of the extracellular nucleic acids present in the antibiotic exposure, but none of the intracellular nucleic acids in the antibiotic exposure.

b. The purpose of putting the filtrates through extraction is to prevent chemical degradation of nucleic acids in the filtrate after collection. DNA Extraction Buffer prevents nucleic acid degradation by digesting and inactivating nuclease proteins. Alternative methods to achieve the same end include other RNA stabilization or nucleic extraction reactions or kits.

c. Centrifugation may be employed instead of a vacuum manifold to speed the filtration.

d. The volume of Lucigen™ DNA Extraction Buffer is chosen so that it is greater than or equal to the volume of filtrate or feed added to it, but not so much larger that the nucleic acids extracted are needlessly diluted. The ratio of DNA Extraction Buffer volume to filtrate or feed volume is preferably within the range of 1 to 10.

e. The volume of antibiotic exposure subjected to filtration was chosen to be larger than the volume transferred to create the feed extraction. This is because it is the concentration of nucleic acid in the filtrate will always be less than or equal to that of the feed. During some nucleic acid quantification techniques, such as any digital nucleic acid amplification, a more accurate concentration of a dilute solution is obtained if one quantifies more volume of the dilute solution. Thus, it is more likely that the filtrate extraction will require more volume to be analyzed.

f. Whenever a volume of the antibiotic exposure is transferred, a small volume ≤10 μL is purposefully not transferred. By not transferring all of the available volume, volumetric pipetting errors are mitigated.

In this experiment, since it is known that each exposure contains adequate numbers of cells of the bacteria of interest, only DNA targets will be quantified. This saves the time and reagents needed to perform reverse transcription of RNA targets.

Next, real-time quantitative PCR (qPCR) is used to quantify the genomic DNA concentrations in each of the filtrate and feed extractions. Specifically, a 3.0 μL volume of each extraction is separately added, according to kit instructions, to 5.0 μL of BioRad SsoFast™ EvaGreen® qPCR 2× Supermix, 1.6 μL of nuclease-free deionized water, and 0.4 μL of a solution of PCR oligonucleotide primers with each primer at a concentration of 10 μM to create a 10 qPCR reaction. There are 192 qPCR reactions in total, and the qPCR supermix, nuclease-free water, and primer solutions can be first mixed together as a master mix before aliquoting 7.0 of the master mix to each well of a 96-well PCR plate, and before addition of the extractions. The primers flank an 80 bp region whose sequence is specific to the Enterobacteriaceae family and common to all of the 23S ribosomal RNA genomic loci in Escherichia coli. The sequences of the primers is as follows: 5′-GGTAGAGCACTGTTTTGGCA-3′ (SEQ ID NO: 11), 5′-TGTCTCCCGTGATAACTTTCTC-3′ (SEQ ID NO: 12). The qPCR reaction is performed on the BioRad CFX96 platform according to manufacturer's instructions. The cycling protocol includes 40 cycles, each comprising a 30 second denaturing step at 95° C., a 60 second annealing step at 60° C., and a 30 second extension step at 60° C.

a. Alternative nucleic acid quantification methods could have been employed, including all of the methods for nucleic acid quantification enumerated earlier in this document. b. The number of qPCR cycles run depends on the user's preferences and can be greater than 40 cycles. There is a tradeoff between a larger dynamic range, longer time to complete the protocol, and the usefulness of high Cq values. Most Cq values above 35 cycles represent non-specific amplification and thus measurement noise.

The output of the qPCR run are the quantification cycles (Cq), which reflect nucleic acid concentration, of the qPCR reaction mixes. To get the extracellular nucleic acid concentration value (NACV) and the total nucleic acid concentration value of a given antibiotic exposure, it is necessary to first calculate the PCR per cycle efficiency, then to convert the Cqs to nucleic acid concentration values, and then finally to normalize the NACVs by the total dilution from the antibiotic exposure. In this experiment, the last step involves dividing the extracellular NACV by

${\left( \frac{60\mu L}{120\mu L} \right)\left( \frac{3\mu L}{10\mu L} \right)} = {{0.1}5}$

and dividing the total NACV by

${\left( \frac{25\mu L}{75\mu L} \right)\left( \frac{3\mu L}{10\mu L} \right)} = {{0.1}{0.}}$

Normalizing must be done in this experiment because a different relative amount of DNA Extraction Buffer was added to the filtrate than to the feed. Dividing the Cq values by a dilution would not yield meaningful values because Cq values exist on a logarithmic scale while the dilutions are on a linear scale. (In a mathematically equivalent alternative, one could define a new quantity equal to the logarithm of the dilution, where the base of the logarithm is the PCR per cycle efficiency, then subtract this new log-scale dilution quantity from the Cq value.)

The “fraction extracellular” (FE) is calculated for each pair of extracellular and total NACVs (normalized for dilutions). The FE is a quantity (i.e. statistic) equal to the dilution normalized extracellular NACV divided by the dilution normalized total NACV. The FE is also a normalized NACV, since it is an NACV divided by a total NACV measurement. The percent extracellular mentioned in Examples 38-42 is the FE multiplied by 100. Calculating the fraction extracellular with NACVs not normalized by total dilution would not yield meaningful results unless the same relative amount of DNA Extraction Buffer was used. Of the 96 FEs calculated above, 8 FEs represent untreated reference conditions and can be called reference FEs.

At this point, one will possess more than one (i.e. 8) reference FEs and more than one treated (i.e. 88) FEs. The treated FEs are further grouped into more than one groups (i.e. the 18 antibiotic compounds) with one continuous-valued predictor variable that was intentionally varied (i.e. the antibiotic concentration) and several other predictor variables that were not varied in this experiment (e.g. exposure duration, initial inoculum). One way to analyze this experiment is to treat all 88 of the treated conditions as separate AST assays with the goal of calling the resistance phenotype of the strain in each condition. Calling resistance phenotypes of each individual AST is a goal that can be framed statistically as a null hypothesis significance testing (NHST) task. Several statistical techniques and frameworks exist that can achieve NHST or classification. A list of appropriate statistical techniques is identifiable by a skilled person upon review of the present disclosure.

To perform NHST and call each individual AST, one can use one of several statistical tests to test the null hypotheses that each treated condition arose from the distribution of reference FEs and represents a resistant phenotype. If the likelihood of generating the observed treated FE is less than a chosen susceptible significance threshold, then one should reject that treated condition's null hypothesis and consider the phenotype of the strain in that condition to be susceptible. One can also define an intermediate significance threshold, so that likelihoods in between the intermediate and susceptible significance thresholds indicate an intermediate resistance phenotype. The distribution of references statistics is not measurable in its entirety, but the 8 reference FEs serve as a sampling of the distribution of reference FEs.

a. The choice of the significance thresholds is subjective, a fundamental reality of statistical philosophy, but it should be chosen a priori (not affected by the measured data) to meet the user's needs. Choosing the significance thresholds involves a tradeoff between the assay's diagnostic sensitivity and specificity. A conventional threshold in research settings is 0.05, but to guard against treatment failure in clinical settings if a resistant strain is called as susceptible, a more stringent susceptible significance threshold such as 0.005, 0.001, 0.0001, or lower can be chosen. Suitable choices for the intermediate significance threshold include 0.05, 0.01, 0.001, or lower, so long as the intermediate significance threshold is not less than or equal to the susceptible significance threshold. b. Because no antibiotic compound and antibiotic concentration pairing (aside from the untreated references) are repeated, only one treated FE is compared against the 8 untreated reference FEs. One can calculate the likelihood of the one treated FE from the distribution assumed for and fitted onto the reference FEs. For example, if one makes the reasonable assumption that the reference FEs are approximately normal, then one can calculate the likelihood of the one treated FE by calculating the z-score of the treated FE. The z-score is calculated by subtracting the sample mean of the reference FEs from the treated FE, then dividing the difference by the sample standard deviation of the reference FEs. Consulting a table of z-scores yields the likelihood. c. If replicates are included, such as by having three separate wells/antibiotic exposures containing 1 μg/mL of ertapenem, then the applicable statistical tests would include any test between two samplings, such as the z-test and its variants, the t-test and its variants, the Wilcoxon-Mann-Whitney test, and the Kolmogorov-Smirnov test. d. Bayesian statistics offers a different framework to NHST than the frequentist methods mentioned in the preceding three paragraphs.

To estimate the strain's MICs for each antibiotic compound tested, one should determine for each compound what is the lowest concentration that leads to a sufficient rate of cell death to cause no net growth of the population at the end of 18 hours. If one only considers the discrete number of concentrations actually tested, then this determination is equivalent to a classification task. Alternatively, one can assume that the MIC is continuous valued and may fall in between the concentrations tested, one may attempt to fit a curve through the treated FEs to interpolate the MIC. This is equivalent to a regression task. Applicable classification and regression algorithms are identifiable by a skilled person upon reading of the present disclosure.

a. A specific, simple algorithm for determining the MIC would be to choose for each of the 18 antibiotic compounds the highest concentration for which our NHST algorithm called the phenotype as susceptible. b. Because the MIC is defined, in its strictest sense, only for the standard broth microdilution AST assay, it may be necessary to find a mathematical function that maps from the FEs found in this assay to the MICs measured by standard broth microdilution. Such a function is commonly found through machine learning techniques as will be understood by a skilled person upon reading of the present disclosure.

In summary, described herein is an antibiotic susceptibility test and related compositions, methods and systems based on detection of a nucleic acid from a target microorganism in a sample in presence or absence of a lysis treatment of the sample.

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compounds, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including webpages patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure, including references cited in any one of the Appendices, are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.

Furthermore, the computer readable form of the sequence listing of the ASCII text file named “P2283-USCIP-2021-06-03-Rep1-Seq-List-2_5 T25.txt”, created on Jun. 3, 2021, and having a file size (not “size on disk”) of 2,802 bytes measured on Windows Server 2016 Standard ver. 1607, is incorporated herein by reference in its entirety.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods may include a large number of optional composition and processing elements and steps.

In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

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1. A method to detect a nucleic acid of a microorganism a sample including the microorganism, the method comprising contacting the sample with an antibiotic to provide an antibiotic treated sample, performing mechanical separation of the microorganism from the nucleic acid in the antibiotic treated sample to provide an antibiotic treated separated sample comprising extracellular nucleic acid of the microorganism, and quantitatively detecting in the antibiotic treated separated sample the extracellular nucleic acid of the microorganism to obtain an detected antibiotic treated nucleic acid concentration value of the microorganism in the antibiotic treated sample, the quantitatively detecting performed in presence of a lysis treatment of the antibiotic treated sample targeting the microorganism,
 2. The method of claim 1, wherein the mechanical separation is performed by filtration or centrifugation.
 3. The method of claim 1, wherein the mechanical separation is performed by filtration and centrifugation.
 4. The method of claim 1, wherein contacting the sample with an antibiotic is performed for up to 90 minutes, or for up to 45 minutes, or for up to 30 minutes, or for up to 15 minutes, or for up to 5 minutes.
 5. The method of claim 1, wherein the quantitatively detecting is performed by performing quantitative real-time PCR (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR).
 6. The method of claim 1, wherein the quantitatively detecting is performed by performing qLAMP, RPA, dLAMP, or dRPA.
 7. The method of claim 1, wherein the detecting is performed by contacting the sample probe specific for a nucleic acid of the microorganism and or for any nucleic acid complementary to the nucleic acid of the microorganism.
 8. The method of claim 1, wherein the sample comprises a plurality of samples and the contacting is performed to provide a plurality of antibiotic treated samples, wherein in each antibiotic treated sample of the plurality of antibiotic treated samples is provided by independently contacting each antibiotic tested sample for a set testing time, and the quantitatively detecting is independently performed following the contacting at a corresponding detection time.
 9. The method of claim 8, wherein the plurality of samples are aliquots provided by partitioning the samples before or after the contacting.
 10. The method of claim 8, wherein the contacting is performed in parallel for each sample of the plurality of samples, and the detecting is performed in parallel for each antibiotic treated sample of the plurality of the antibiotic treated sample.
 11. The method of claim 1, wherein the method further comprises concurrently or after the contacting and before the detecting: performing an enhancing treatment of the antibiotic treated sample.
 12. The method of claim 1, the method further comprising detecting a nucleic acid concentration ratio in the sample by comparing the detected antibiotic treated nucleic acid concentration value with a detected reference nucleic acid concentration value of a nucleic acid concentration parameter of the microorganism in the sample.
 13. The method of claim 12, wherein the reference nucleic acid concentration value of the nucleic acid concentration parameter is a control nucleic acid concentration value obtained by detecting a nucleic acid concentration for the nucleic acid of the microorganism in a control sample of the isolate or specimen comprising the microorganism, the detecting in the control sample performed in presence of a lysis treatment of the control sample following pre-lysis separation of microorganism from the nucleic acid in the control sample to provide a control separate sample comprising extracellular nucleic acid of the microorganism, the detecting a nucleic acid concentration in the control sample performed to provide the control nucleic acid concentration value for the nucleic acid of the microorganism.
 14. The method of claim 13, wherein the control sample is an aliquot of the sample obtained by partitioning the sample before the contacting.
 15. The method of claim 12, wherein the reference nucleic acid concentration value is a total nucleic acid concentration value obtained by detecting the total nucleic acid concentration of the microorganism in a reference sample of an isolate or specimen comprising the microorganism and either treated or not treated with the antibiotic, to provide the total nucleic acid concentration value for the nucleic acid of the microorganism.
 16. The method of claim 15, wherein the reference sample is sample is an aliquot of the sample obtained by partitioning the sample before and/or after the contacting.
 17. The method of claim 12, wherein the reference nucleic acid concentration value of the nucleic acid concentration parameter is a control nucleic acid concentration and wherein the nucleic acid concentration ratio is more than 2 or less than 0.5.
 18. The method of claim 12, wherein the duration of the contacting of the sample with an antibiotic is 20 minutes or less, wherein the nucleic acid concentration ratio is more than 2 or less than 0.5; more than 3 or less than 0.33; more than 5 or less than 0.2; more than 10 or less than 0.1, more than 15 or less than 0.067, more than 20 or less than 0.05.
 19. The method of claim 18, wherein the nucleic acid is DNA and/or RNA.
 20. The method of claim 12, wherein the contacting of the sample with an antibiotic is performed for 30 minutes or less, and wherein the nucleic acid concentration ratio is more than 3 or less than 0.33; more than 5 or less than 0.2; more than 10 or less than 0.1, more than 15 or less than 0.067, more than 20 or less than 0.05.
 21. The method of claim 20 wherein the nucleic acid is DNA and/or RNA.
 22. The method of claim 12, wherein the nucleic acid concentration ratio is indicative of resistance or susceptibility of the microorganism to the antibiotic.
 23. The method of claim 22, wherein the method further comprises establishing susceptibility of the microorganism to the antibiotic when the nucleic acid concentration ratio is above an accessible threshold of susceptibility.
 24. The method of claim 22, wherein the method further comprises establishing resistance of the microorganism to the antibiotic when the nucleic acid concentration ratio is below an accessible threshold of resistance.
 25. The method of claim 1, wherein the method further comprises purifying or extracting the nucleic acid from the antibiotic treated sample before the detecting.
 26. The method of claim 25, wherein the method further comprises extracting the nucleic acid from the antibiotic treated sample concurrently or after the lysis treatment and before the detecting.
 27. The method of claim 1, wherein the antibiotic is a beta-lactam or a carbapenem.
 28. The method of claim 1, wherein the microorganism belongs to the family Enterobacteriaceae.
 29. The method of claim 1, wherein the sample is obtained from a specimen pretreated to enrich the sample with nucleic acids or with the target microorganism, and/or to remove human nucleic acid, nucleic of other microorganisms.
 30. The method of claim 1, wherein the sample is urine sample. 